Multimeric oligonucleotide compounds

ABSTRACT

The disclosure provides multimeric oligonucleotide compounds, comprising two or more target-specific oligonucleotides (e.g., antisense oligonucleotides (ASOs)), each being resistant to cleavage, and linked together by a cleavable linker. In particular, two or more linked target-specific oligonucleotides, each to a different target, allows concomitant inhibition of multiple genes&#39; expression levels, while exhibiting favorable pharmacokinetic and pharmacodynamic properties. Methods of making and uses of the described compounds are also provided

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 to U.S.provisional patent application, U.S. Ser. No. 61/534,561, filed Sep. 14,2011, entitled “Multimeric Antisense Oligonucleotides,” the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to oligonucleotide reagents, oligonucleotidetherapeutics, and methods of making and using thereof.

BACKGROUND OF THE INVENTION

The development of oligonucleotides into clinical medicines and theiruse as basic research tools is an ongoing endeavor. For example, the useof antisense oligonucleotides for gene silencing was described as earlyas 1978. Since this time other oligonucleotide based approaches haveemerged for regulating gene expression, including RNA interference,microRNAs, and, recently, targeted inhibition or inactivation of longnon-coding RNAs.

Although natural phosphodiester-backbone oligonucleotides are taken upby cells efficiently, they are highly susceptible to nucleasedegradation in plasma, which limits their effectiveness as therapeuticsin some cases. In some instances, therefore, it is advantageous to limitor control the extent to which oligonucleotides are degraded bynucleases. In this regard, a number of modified nucleotides (e.g., LNAs)and backbone modifications (e.g., phosphorothioates, methylphosphonates)have been reported that improve stability in some instances.Nonetheless, it remains as current objective in oligonucleotide basedresearch and development to obtain oligonucleotides having favorablepharmacokinetic and pharmacodynamic properties.

SUMMARY OF THE INVENTION

According to some aspects of the invention, multimeric oligonucleotidecompounds are provided that are useful for regulating gene expressionand function. Some aspects of the invention are based on the discoverythat relatively high levels of a monomeric oligonucleotides can beachieved in a target tissue or cell when monomeric units are connectedby a cleavable linker (e.g., an endonuclease-sensitive linker) andadministered as a multimer. In some embodiments, the properties of alinker are selected to modulate the pharmacokinetic and pharmacodynamicproperties of the multimeric oligonucleotide compounds. For example, insome embodiments, linker properties can be tuned to control the extentto which monomeric units are released in a particular tissue-type orcell-type to be targeted.

In some embodiments, an advantage of using multimers is that it allowssimultaneous knockdown of multiple targets, while exploiting thepharmacokinetic and/or pharmacodynamic advantages of the administeredoligonucleotide. In some embodiments, a sequence-specific concomitantknockdown of two or more targets may be achieved with a heteromultimercontaining targeting oligonucleotides directed against several targetgene combinations.

In some embodiments, multimeric oligonucleotide compounds providedherein comprise two or more targeting oligonucleotides linked togetherby a cleavable linker. In some embodiments, each targetingoligonucleotide has a region complementary to a target region of agenomic target sequence. In some embodiments, the targetingoligonucleotides hybridize to a target nucleic acid encoded by a genomictarget sequence and inhibit the function and/or effect degradation ofthe target nucleic acid. The target nucleic acid may be, for example, along non-coding RNA (lncRNA), microRNA, or mRNA.

In some embodiments, the targeting oligonucleotide is an antisenseoligonucleotide (ASO), siRNA (e.g., a single stranded siRNA), miRNAsponge, or anti-microRNA antisense oligonucleotide (AMO). In someembodiments, the targeting oligonucleotide binds specifically to atarget nucleic acid in a cell and brings about degradation of the targetnucleic acid. In some embodiments, the degradation is mediated by RNAseH. In some embodiments, the degradation is mediated by an RNAi pathway.In some embodiments, the targeting oligonucleotide binds specifically toits target nucleic acid in a cell and inhibits the function of thetarget nucleic acid. For example, in some embodiments, the targetingoligonucleotide binds to a target lncRNA and inhibits interaction of thelncRNA with one or more interacting proteins (e.g., a subunit ofPolycomb Repressor Complex 2 (PRC2)).

According to some aspects of the invention, compounds are provided thatcomprise the general formula: X-L-[X-L]_(i)-X, in which i is an integerfrom 0 to 9, the value of which indicates the number of units of[X-L]_(i) present in the compound, in which each X is independently atargeting oligonucleotide having a region of complementarity comprisingat least 7 contiguous nucleotides complementary to a target region of agenomic target sequence, and each L is a linker that links at least twoXs and that is more susceptible to cleavage in a mammalian extract thaneach X. In some embodiments, when i=0, and the general formula is5′X3′-L-5′X3′ and when the target regions complementary to the first Xand second X do not overlap in the genomic target sequence, the 5′-endof the target region complementary to the first X and the 3′-end of thetarget region complementary to the second X are not within a distance of0 to 4 nucleotides in the genomic target sequence. In some embodiments,the 5′-end of the target region complementary to the first X and the3′-end of the target region complementary to the second X are not withina distance of 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0to 8, 0 to 9, 0 to 10, 0 to 15, 0 to 20, 0 to 25 or more nucleotides inthe genomic target sequence. In some embodiments, the targetingoligonucleotides are 8 to 15, 10 to 16, 10 to 20, 10 to 25, 15 to 30, 8to 50, 10 to 100 or more nucleotides in length. In some embodiments, thetargeting oligonucleotides are 8, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides in length.

In some embodiments, at least one L does not comprise an oligonucleotidehaving a self-complementary nucleotide sequence. In some embodiments,all Ls do not comprise an oligonucleotide having a self-complementarynucleotide sequence. In some embodiments, at least one L does notcomprise an oligonucleotide having a nucleotide sequence that iscomplementary to a region of the genomic target sequence that iscontiguous with the target regions complementary to two immediatelyflanking Xs of the at least one L. In some embodiments, the compounddoes not comprise a ribozyme. In some embodiments, all Ls do notcomprise an oligonucleotide having a nucleotide sequence that iscomplementary to a region of the genomic target sequence that iscontiguous with the target regions complementary to two immediatelyflanking Xs.

In some embodiments, i is an integer from 0 to 3, 1 to 3, 1 to 5, 1 to9, 1 to 15, 1 to 20. In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20 or more. In some embodiments, the at least one L linkercomprises an oligonucleotide that is more susceptible to cleavage by anendonuclease in the mammalian extract than the targetingoligonucleotides. In certain embodiments, at least one L is a linkerhaving a nucleotide sequence comprising from 1 to 10 thymidines oruridines. In some embodiments, at least one L is a linker having anucleotide sequence comprising deoxyribonucleotides linked throughphosphodiester internucleotide linkages. In certain embodiments, atleast one L is a linker having a nucleotide sequence comprising from 1to 10 thymidines linked through phosphodiester internucleotide linkages.In some embodiments, at least one L is a linker having a nucleotidesequence comprising from 1 to 10 uridines linked throughphosphorothioate internucleotide linkages. In certain embodiments, atleast one L is a linker having the formula:

in which Z is an oligonucleotide. In some embodiments, Z has anucleotide sequence comprising from 1 to 10 thymidines or uridines. Incertain embodiments, at least one L does not comprise an oligonucleotidehaving a self-complementary nucleotide sequence and does not comprise anoligonucleotide having a nucleotide sequence that is complementary to aregion of the genomic target sequence that is contiguous with twoflanking target regions. In some embodiments, at least one L is a linkerthat does not comprise an oligonucleotide having an abasic site.

In certain embodiments, for at least one L, the linker comprises apolypeptide that is more susceptible to cleavage by an endopeptidase inthe mammalian extract than the targeting oligonucleotides. In someembodiments, the endopeptidase is trypsin, chymotrypsin, elastase,thermolysin, pepsin, or endopeptidase V8. In some embodiments, theendopeptidase is cathepsin B, cathepsin D, cathepsin L, cathepsin C,papain, cathepsin S or endosomal acidic insulinase. In certainembodiments, at least one L is a linker comprising a peptide having anamino acid sequence selected from: ALAL (SEQ ID NO: 125), APISFFELG (SEQID NO: 126), FL, GFN, R/KXX, GRWHTVGLRWE (SEQ ID NO: 127), YL, GF, andFF, in which X is any amino acid.

In some embodiments, at least one L is a linker comprising the formula—(CH₂)_(n)S—S(CH₂)_(m)—, wherein n and m are independently integers from0 to 10. In certain embodiments, at least one L the linker comprises alow pH-labile bond. In some embodiments, the low pH-labile bondcomprises an amine, an imine, an ester, a benzoic imine, an amino ester,a diortho ester, a polyphosphoester, a polyphosphazene, an acetal, avinyl ether, a hydrazone, an azidomethyl-methylmaleic anhydride, athiopropionate, a masked endosomolytic agent or a citraconyl group.

In some embodiments, at least one L is a branched linker. In certainembodiments, the branched linker comprises a phosphoramidite linkage. Incertain embodiments, the compound is a non-symmetrical branched trimer.In certain embodiments, the compound is a symmetrical branched trimer.In some embodiments, at least one L is a linker that is at least 2-foldmore sensitive to cleavage in the presence of a mammalian extract thanthe targeting oligonucleotides.

In some embodiments, the compound may have the following generalformula:

in which i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, in which j and kare independently 0 or 1, the value of which indicates, respectively,the number of X_(j) and X_(k) present, and in which l and m areindependently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, the value ofwhich indicates, respectively, the number of units of [X-L]_(l) and[L-X]_(m) present in the compound. In some embodiments, at least one of[X-L]_(l) and [L-X]_(m) are present.

In some embodiments, the compound has the following general formula:X-L-[X-L]_(i)-X. In some embodiments, the compound has the followinggeneral formula:

In some embodiments, the compound has the following general formula:

in which j and k are independently 0 or 1, the value of which indicates,respectively, the number of X_(j) and X_(k) present in the compound, andat least one of X_(j) and X_(k) are present in the compound.

According to some aspects of the invention, compounds are provided thatcomprise at least two targeting oligonucleotides linked through a linkerthat is at least 2-fold more sensitive to enzymatic cleavage in thepresence of a mammalian extract than the at least two targetingoligonucleotides, wherein each targeting oligonucleotide has a region ofcomplementarity comprising at least 7 contiguous nucleotidescomplementary to a target region of a genomic target sequence. In someembodiments, the targeting oligonucleotides are 8 to 15, 10 to 16, 12 to16, 10 to 20, 10 to 25, 15 to 30, 8 to 50, 10 to 100 or more nucleotidesin length. In some embodiments, the targeting oligonucleotides are 8,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40,50, 60, 70, 80, 90, 100 or more nucleotides in length.

In some embodiments, the linker is at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or moresensitive to enzymatic cleavage in the presence of a mammalian extractthan the two targeting oligonucleotides. In some embodiments, the linkeris an oligonucleotide. In some embodiments, the oligonucleotide has asequence that is not complementary to the genomic target sequence at aposition immediately adjacent to the target region. In certainembodiments, the mammalian extract is an extract from kidney, liver,intestinal or tumor tissue. In some embodiments, the mammalian extractis a cell extract. In some embodiments, the mammalian extract is anendosomal extract.

In certain embodiments, at least one targeting oligonucleotide comprisesat least one ribonucleotide, at least one deoxyribonucleotide, or atleast one bridged nucleotide. In some embodiments, the bridgednucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modifiednucleotide. In some embodiments, at least one targeting oligonucleotidecomprises at least one a 2′-fluoro-deoxyribonucleotide. In someembodiments, at least one targeting oligonucleotide comprisesdeoxyribonucleotides flanked by at least one bridged nucleotide on eachof the 5′ and 3′ ends of the deoxyribonucleotides. In some embodiments,at least one targeting oligonucleotide comprises phosphorothioateinternucleotide linkages between at least two nucleotides. In certainembodiments, at least one targeting oligonucleotide comprises a 2′O-methyl. In some embodiments, at least one targeting oligonucleotidecomprises a G-clamp, 5-propynyl, or 5-octadienyl-pyrimidine. In certainembodiments, at least one targeting oligonucleotide is a gapmercomprising RNase H recruiting nucleotides. In some embodiments, at leastone targeting oligonucleotide is a single stranded siRNA.

In certain embodiments, the compound is linked to a functional moiety(e.g., a lipophilic moiety or targeting moiety that binds to a cellsurface receptor). In some embodiments, the functional moiety is linkedto a targeting oligonucleotide. In some embodiments, the functionalmoiety is linked to a linker.

In certain embodiments, at least two targeting oligonucleotides are inthe same 5′ to 3′ orientation relative to the linker. In someembodiments, at least two targeting oligonucleotides are in opposite 5′to 3′ orientations relative to the linker. In certain embodiments, atleast one targeting oligonucleotide is linked to the linker through aterminal nucleotide. In certain embodiments, at least one targetingoligonucleotide is linked to the linker through an internal nucleotide.In some embodiments, at least one targeting oligonucleotide is asingle-stranded oligonucleotide.

In certain embodiments, the target region complementary to at least onetargeting oligonucleotide is present in the sense strand of a gene. Insome embodiments, the gene is an non-coding RNA gene. In certainembodiments, the non-coding RNA gene is a long non-coding RNA gene. Insome embodiments, the non-coding RNA gene is an miRNA gene. In someembodiments, the gene is a protein coding gene. In certain embodiments,the genomic target sequence of at least one targeting oligonucleotide isthe sequence of a PRC-2 associated region. In certain embodiments, atleast two target regions are present in the sense strand of differentgenes. In certain embodiments, at least two target regions are presentin the sense strand of the same gene. In some embodiments, at least twotarget regions are different. In some embodiments, at least two targetregions are identical. In certain embodiments, the product of the genemediates gene expression through an epigenetic mechanism.

According to some aspects of the invention, compositions are providedthat comprise any of the compounds disclosed herein and a carrier. Insome embodiments, the compositions comprise a buffered solution. In someembodiments, the compound is conjugated to the carrier. According tosome aspects of the invention, pharmaceutical compositions are providedthat comprise any of the compounds disclosed herein and apharmaceutically acceptable carrier. In some embodiments, kits areprovided that comprise a container housing any of the compounds orcompositions disclosed herein.

According to some aspects of the invention, methods of increasingexpression of a target gene in a cell are provided. In some embodiments,the methods comprise: contacting the cell with any of the compoundsdisclosed herein, and maintaining the cell under conditions in which thecompound enters into the cell. In some embodiments of the methods, thegenomic target sequence of at least one targeting oligonucleotide of thecompound is present in the sense strand of an lncRNA gene, the productof which is an lncRNA that inhibits expression of the target gene. Insome embodiments, presence of the compound in the cell results in alevel of expression of the target gene that is at least 50% greater, atleast 60% greater, at least 70% greater, at least 80%, or at least 90%greater than a level of expression of the target gene in a control cellthat does not contain the compound.

According to some aspects of the invention, methods of increasing levelsof a target gene in a subject are provided. In some embodiments, themethods comprise administering any of the compounds disclosed herein tothe subject. In some embodiments, the genomic target sequence of atleast one targeting oligonucleotide of the compound is present in thesense strand of an lncRNA gene, the product of which inhibits expressionof the target gene.

According to some aspects of the invention, methods of treating acondition associated with altered levels of expression of a target genein a subject are provided. In some embodiments, the condition isassociated with decreased or increased levels of expression of thetarget gene compared to a control subject who does not have thecondition. In some embodiments, the methods comprise administering thecompound to the subject. In some embodiments, the genomic targetsequence of at least one targeting oligonucleotide of the compound ispresent in the sense strand of an lncRNA gene, the product of whichinhibits expression of the target gene. Accordingly, in someembodiments, the at least one targeting oligonucleotide hybridizes tothe lncRNA and inhibits its function or brings about its degradation.

According to some aspects of the invention, methods of modulatingactivity of a target gene in a cell are provided. In some embodiments,the methods comprise contacting the cell with any of the compoundsdisclosed herein, and maintaining the cell under conditions in which thecompound enters into the cell. In some embodiments, presence of thecompound in the cell results in reduced expression or activity of thetarget gene in the cell. According to some aspects of the invention,methods of modulating levels of a target gene in a subject are provided.In some embodiments, the methods comprise administering any of thecompounds disclosed herein to the subject. In some embodiments thegenomic target sequence of at least one targeting oligonucleotide ispresent in the sense strand of the target gene. In some embodiments, thetarget gene is a protein coding gene or non-coding gene.

In some embodiments, multimeric oligonucleotide compounds are providedthat comprise two or more targeting oligonucleotides (e.g., ASOs), eachhaving a nuclease-resistant modified backbone, wherein the targetingoligonucleotides are linked to each other by one or more degradablelinkers. In some embodiments, the backbone contains inter-nucleosidelinkages. In some embodiments, the individual linked targetingoligonucleotides, contained in a compound, may be directed to the sametarget, or to multiple targets. The multimeric compounds can behomodimers, homotrimers, etc., heterodimers, heterotrimers, etc. Theycan be linear, branched, or circular.

In some embodiments, the invention is based, in part, on the discoverythat multimeric oligonucleotide compounds (e.g., a 14-mer ASO linked toanother 14-mer ASO) show significantly higher levels of thecorresponding monomeric oligonucleotide compounds in the liver when themonomer units are connected by a rapidly degradable linker (e.g., anuclease-sensitive linker or a disulfide linker), as opposed to a linkerthat is nuclease-resistant and, therefore, slowly degradable.Unexpectedly, the detected liver levels of the dimer-derived monomericunits were five to ten times higher than that of the correspondingmonomers administered in the monomeric form. The increased delivery tothe liver was also associated with a more effective target mRNAknockdown after 14 days of dosing in mice. The invention is therefore,in part, based on the realization that the type and properties of thelinker can thus be used to modulate the pharmacokinetic andpharmacodynamic properties of the dimer antisense molecules. In someembodiments, rapidly degradable linkers are referred as “cleavable”(such as, e.g., a nuclease-sensitive, phosphodiester, linkage or alinker comprising a disulfide bond), while more stable linkages, suchas, e.g., nuclease-resistant phosphorothioates, as referred to as“noncleavable.”

In illustrative embodiments, the compounds are directed to one or morehepatic targets ASOs are directed to hepatic targets, including but notlimited to ApoC3 and ApoB.

In some embodiments, targeting oligonucleotides (e.g., ASOs) contain 12to 16 nucleotide bases, wherein one or more targeting oligonucleotidesare gapmers. Targeting oligonucleotides (e.g., ASOs), including gapmers,can comprise a 2′ modification in the sugar residues (e.g.,locked-nucleic acid (LNA) modification), 2′-O-methyl and 2′-fluoromodification, and/or a nucleotide modification such as G-clamp,5-propynyl, and 5-octadienyl-pyrimidine.

The invention further provides pharmaceutical compositions, comprisingcompounds of the invention along with pharmaceutically acceptableexcipients. In certain embodiments, the pharmaceutical composition ischaracterized by one or more of the following properties whenadministered in vivo:

(a) increased concentration in the liver and reduced clearance bykidneys as compared to respective monomeric targeting oligonucleotides(e.g., ASOs);

(b) longer duration of target knockdown as compared to respectivemonomeric targeting oligonucleotides (e.g., ASOs); and

(c) lower effective concentrations as compared to respective monomerictargeting oligonucleotides (e.g., ASOs) and/or the same multimericoligonucleotide compound, wherein the cleavable linker is substitutedwith a noncleavable linker.

The invention further provides methods of inhibiting mRNA levels of oneor more targets, comprising administering to a cell or a subject thecompound of the invention in an amount effective to inhibit theexpression of the target(s). In some embodiments, the methods provide atherapeutically effective knockdown of the target(s) persists for twoweeks or longer following the administration. The method can be usedwith targets that are associated with a metabolic disease, cancer,cardiovascular disease, and other conditions.

The foregoing and following descriptions are illustrative andexplanatory only and are not restrictive of the invention, as claimed inthis text, the multimeric targeting oligonucleotides (e.g., ASOs) may bereferred to by the respective target names only, e.g., “ApoC3-ApoC3dimer” stands as a short hand for “ApoC3-ApoC3 ASO dimer.”

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a schematic representation of an exemplary construct, inwhich two 14-mer gapmers (e.g., 3LNA-8DNA-3LNA as illustrated) areconnected via a linker (represented light shaded circles). FIG. 1B showsexamples of various configurations of dimers and multimers (homopolymersor heteropolymers). FIGS. 1C and 1D show details of the chemicalstructures of certain multimeric ASOs.

FIGS. 2A-2D demonstrate in vitro stability of dimers in plasmas andtheir degradation in liver homogenates, as determined by liquidchromatography-mass spectrometry (LC-MS). FIGS. 2A and 2B demonstrateslow degradation of both ApoC3 ASO monomer (SEQ ID NO:1, designated asper Example 2(E)) and cleavable ApoC3-ApoC3 ASO dimers (SEQ ID NO:2 andSEQ ID NO:4) in murine and monkey plasmas respectively. FIG. 2Cdemonstrates efficient cleavage into monomers of the cleavableApoC3-ApoC3 ASO dimers (SEQ ID NO:2 and SEQ ID NO:4) and the relativestability ApoC3 ASO monomer (SEQ ID NO:1) in mouse liver homogenate.FIG. 2D shows cleavable SEQ ID NO:18) and noncleavable SEQ ID NO: 19)ApoB-ApoB ASO homodimers incubated in murine plasma or liver homogenate,demonstrating stability of both types of molecules in plasma, and a moreefficient cleavage into monomers of the cleavable version in the liverhomogenate.

FIGS. 3A-3K address various aspects of linker designs in homodimers. Forthe results shown in FIGS. 3A, 3B and 3D, Hep3B cells were treated atvarious concentrations (0.001, 0.006, 0.03, 0.2, 0.8, 4.0, 20 and 100nM) of the indicated oligonucleotides formulated with a lipotransfectionagent. mRNA content and cell viability was determined 48 hours aftertreatment. For the results shown in FIGS. 3C and 3E-3K, Hep3B cells weretreated at eight concentrations (0.1, 0.6, 3.0, 20, 80, 400, 2000 and10,000 nM) of the indicated oligonucleotides without any transfectionagent (“gymnotic delivery”). mRNA content and cell viability weredetermined after 8 days of treatment. In all cases, the graphs depictpercentage effect relative to a non-specific oligonucleotide (negativecontrol).

FIGS. 4A-4M address various aspects of the design of variousheterodimers (di- and trimers). For the results shown in FIG. 4A, Hep3Bcells were treated at various concentrations (0.001, 0.006, 0.03, 0.2,0.8, 4.0, 20 and 100 nM) of the indicated oligonucleotides formulatedwith a lipotransfection agent. mRNA content and cell viability weredetermined 48 hours after treatment. For the results shown in FIGS.4B-4M, Hep3B cells were treated at eight concentrations (0.1, 0.6, 3.0,20, 80, 400, 2000 and 10,000 nM) of the indicated oligonucleotideswithout any transfection agent (“gymnotic delivery”). mRNA content andcell viability were determined after 8 days of treatment. In all cases,the graphs depict percentage effect relative to a non-specificoligonucleotide (negative control).

FIGS. 5A-5C demonstrate that under the conditions tested, the timecourse of knock-down depended on the type of linker used to connect thetwo antisense moieties in the dimeric ASOs. Human ApoC3 transgenic micewere administered a single subcutaneous dose of homodimers SEQ ID NO:5or 3 (which are disulphide-linked homodimers of the same monomer) at 10mg/kg, or vehicle. FIG. 5A demonstrates an associated increasedreduction of the liver ApoC3 mRNA levels in human ApoC3 transgenic micefollowing treatment with the endonuclease-sensitive,phosphodiester-linked, homodimers (SEQ ID NO:4 and SEQ ID NO:2).Homodimers SEQ ID NO:4 and 2 exhibited an increased reduction of liverApoC3 mRNA levels compared to the monomer (SEQ ID NO:1) after 14 days.

FIGS. 5B and 5C show ApoC3 protein knockdown 7 days (FIG. 5B) and 14days (FIG. 5C) after a single 10 mg/kg dose of the SEQ ID NO: 1 monomerand dimeric LNA gapmers SEQ ID NO:2-SEQ ID NO:5 in human ApoC3transgenic mice. The figures demonstrate increased duration in thereduction of serum ApoC3 protein levels in human ApoC3 transgenic micefollowing treatment with the endonuclease-sensitivephosphodiester-linked homodimers, SEQ ID NO:1, SEQ ID NO:4 and SEQ IDNO:2. Homodimers SEQ ID NO:4 and SEQ ID NO:2 exhibited a reduction ofserum ApoC3 levels similar to monomer SEQ ID NO: 1 after 7 days, but incontrast to the monomer, the reduction the reduction in target geneexpression in cells treated with the cleavable dimers (SEQ ID NO:2 or 4)was sustained and, as a result, increased compared to SEQ ID NO:1 after14 days.

FIGS. 6A-6C show illustrative LC-MS results for samples extracted fromliver for the following ASOs respectively SEQ ID NO:2 (FIG. 6A), SEQ IDNO:3 (FIG. 6B), and SEQ ID NO:4 (FIG. 6C). “IS” designates an internalstandard.

FIGS. 7A and 7B illustrate that SEQ ID NO: 21, an ApoC3/ApoB heterodimerASO with an endonuclease sensitive phosphodiester linker, significantlydown-regulated liver expression of both target mRNAs [i.e, human APOC3(FIG. 7A) and mouse ApoB (FIG. 7B)].

FIGS. 8A and 8B illustrate the effects of these treatments on in vivotarget mRNAs in the liver. Data in these figures are plotted as %knockdown of the target mRNAs with knockdown of mouse apoB mRNA plottedon the x axis and knockdown of human ApoC3 (i.e., the transgene) plottedon the y axis.

FIGS. 9A and 9B illustrate differences in concentrations of ApoB monomerafter overnight incubation at 37° C. or under frozen conditions ofheterodimers and ApoB monomer ASOs in liver and kidney homogenates. BLQis “Beneath Limit of Quantification.”

FIG. 10 illustrate differences in concentrations of ApoB monomerdetected in plasma 3 days post-treatment with heterodimers and ApoBmonomer ASOs.

FIGS. 11A and 11B illustrate measured concentrations of ApoB monomermetabolite in kidneys at Day 3 and Day 14 following administration ofheterodimers and ApoB monomer ASOs.

FIGS. 12A and 12B illustrate measured concentrations of ApoB monomermetabolite in liver at Day 3 and Day 14 following administration ofheterodimers and ApoB monomer ASOs.

FIGS. 13A and 13B illustrate that dimer oligonucleotides significantlydecreased miR-122 (10 mg/kg dose, mouse liver).

FIGS. 14A and 14B illustrate that dimer oligonucleotides significantlydecreased miR-122 (50 mg/kg dose, mouse liver).

FIG. 15 illustrates that dimer oligonucleotides are ˜5× more active thanmonomer (in vivo 7 d study).

FIGS. 16A, 16B, and 16C illustrate that dimer oligonucleotides robustlydecreased Malat-1 lncRNA expression.

Unless otherwise stated, the numbers in the figures with hash signs(such as #1, #50, etc.) correspond to the respective SEQ ID NOs as perTable 1.

DETAILED DESCRIPTION OF THE INVENTION

Multimeric oligonucleotide compounds are provided that are useful forregulating gene expression and/or function. In general, the multimericoligonucleotide compounds provided herein comprise two or more targetingoligonucleotides linked together by a cleavable linker. The multimericoligonucleotides are useful for regulating the expression or function ofa wide range of target nucleic acids including, for example, a longnon-coding RNA (lncRNA), microRNA, or mRNA. In some embodiments, thetargeting oligonucleotide of the multimer is an antisenseoligonucleotide (ASO), siRNA (e.g., a single stranded siRNA), miRNAsponge, or anti-microRNA antisense oligonucleotide (AMO). However, othertypes of targeting oligonucleotides may be used.

A. General Structure of Multimeric Oligonucleotides

Multimeric oligonucleotide compounds are provided that comprise thegeneral formula: X-L-[X-L]_(i)-X, in which i is an integer, the value ofwhich indicates the number of units of [X-L]_(i) present in thecompound, and in which each X is a targeting oligonucleotide and each Lis a linker that links at least two Xs and that is more susceptible tocleavage in a mammalian extract than each X. In some embodiments, i is0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more,

As used herein, the term “mammalian extract” refers to a sampleextracted from a mammalian tissue, cell or subcellular compartment(e.g., an endosome). Generally, a mammalian extract comprises one ormore biomolecules (e.g., enzymes) from the tissue, cell or subcellularcompartment. In some embodiments, a mammalian extract comprises one ormore of a nuclease, peptidase, protease, phosphatase, oxidase, andreductase. The mammalian extract may be an extract from any tissue,including, for example, kidney, liver, intestinal or tumor tissue. Themammalian extract may be a cell extract or an extract from a subcellularcomponent, such as a nuclear extract, or an endosomal extract.

As used herein, the term “cleavage” refers to the breaking of one ormore chemical bonds in a relatively large molecule in a manner thatproduces two or more relatively small molecules. Cleavage in themammalian extract may be mediated by a nuclease, peptidase, protease,phosphatase, oxidase, or reductase, for example. In some embodiments,the term “cleavable,” as used herein, refers to rapidly degradablelinkers, such as, e.g., phosphodiester and disulfides, while the term“noncleavable” refer to more stable linkages, such as, e.g.,nuclease-resistant phosphorothioates (e.g., a racemic mixture of Sp andRp diastereoisomers, as used in the Examples below, or a backboneenriched in Sp form). The properties of cleavable and noncleavablelinkers are described in further detail herein.

In one example, the compound has the following general formula:

In this formula, i is an integer indicating the number of units of[X-L]_(i) present in the compound; j and k are independently 0 or 1, thevalue of which indicates, respectively, the number of X_(j) and X_(k)present in the compound; and l and m are integers the value of whichindicate, respectively, the number of units of [X-L]_(l) and [L-X]_(m)present in the compound. In some embodiments, i is 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20 or more. In certain embodiments, l and m areindependently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more. Incertain embodiments, at least one of [X-L]_(l) and [L-X]_(m) are presentin the compound. In some embodiments, i, j, k, l, and m are 0. In someembodiments, i is 1, and j, k, l, and m are 0.

In one example, the compound may have the following general formula:X-L-[X-L]_(i)-X, in which i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In another example, the compound may have the following general formula:

in which i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In another example, the compound may have the following general formula:

in which j and k are independently 0 or 1, the value of which indicates,respectively, the number of X_(j) and X_(k) present, and at least one ofX_(j) and X_(k) are present in the compound.

Typically, the targeting oligonucleotide has a region of complementaritycomprising at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 15, or at least 20 contiguousnucleotides complementary to a target region of a genomic targetsequence. The targeting oligonucleotide may have a region ofcomplementarity comprising 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 45, or 50 contiguous nucleotides complementaryto a target region of a genomic target sequence. It should beappreciated that, in some embodiments, the region of complementary mayhave one or more mismatches compared with the nucleotide sequence of thetarget region provided that the targeting oligonucleotide is stillcapable of hybridizing with the target region. In some embodiments, theregion of complementary has no mismatches compared with the nucleotidesequence of the target region. It should also be appreciated that atargeting oligonucleotide may hybridize with a target region throughWatson-Crick base pairing, Hoogsteen base pairing, reverse-Hoogsteenbinding, or other binding mechanism. In some embodiments, the targetingoligonucleotide is an aptamer, e.g., an aptamer that binds to anintracellular or nuclear protein.

In some multimeric oligonucleotides, for two Xs, a first X and a secondX, that are separated by a single L, the 5′-end of the target regioncomplementary to the first X and the 3′-end of the target regioncomplementary to the second X are not within a distance of 0 to 1, 0 to2, 0 to 3, 0 to 4, 0 to 5, 0 to 10, 0 to 15, 0 to 20, 0 to 25, 0 to 50,nucleotides in the genomic target sequence when the target regionscomplementary to the first X and second X do not overlap in the genomictarget sequence. In some instances the different X's havecomplementarity to the same target and in other instances to differenttarget. When the X's have complementarity to the same target the nucleicacid sequence of the X's may be identical with one another oroverlapping or completely distinct.

In some embodiments, multimeric oligonucleotide compounds comprisesASOs. The invention provides in some embodiments multimericoligonucleotide compounds, comprising two or more target-specificantisense oligonucleotides (ASOs), each ASO having a nuclease-resistantmodified backbone, in which the targeting oligonucleotides are linked toeach other by one or more degradable linkers. The term “monomeric” or“monomer,” in the context of targeting oligonucleotides (e.g., ASOs),refers to an targeting oligonucleotide that (i) is directed to a singlesite or a single contiguous stretch of nucleotides on a target and (ii)is not covalently linked to the another targeting oligonucleotidedirected to the same or another site on the same or another target.Multimeric oligonucleotide compounds are not monomeric because theycontain targeting oligonucleotides (e.g., ASOs) that are covalentlylinked to each other.

The number of targeting oligonucleotides (e.g., ASOs) in a multimericoligonucleotide compound of the invention may be two or more, three ormore, four or more, etc. For example, a multimeric oligonucleotidecompound may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more individualTargeting oligonucleotides (e.g., ASOs) directed to one or more targets.The individual Targeting oligonucleotides (e.g., ASOs) can be specificto the same or different targets. For example, as illustrated in FIG.1A, in some embodiments, the targeting oligonucleotide is a dimercomprising two targeting oligonucleotides specific to the same target,or a dimer comprising two targeting oligonucleotides specific to twodifferent targets, or alternatively, a trimer comprising three targetingoligonucleotides specific to the same target, or a trimer comprisingthree targeting oligonucleotides specific three different targets, etc.In some cases, the individual targeting oligonucleotides can be specificto the same target, yet directed to distinct target sites on the target,such as two sites on the target sequence that are separated by at least10, 20, 50, 100, 300 or more nucleotides. In some embodiments, thetarget sites can be directly adjacent to each other and not separated byany intervening sequences.

As shown in FIG. 1B, the multimers can be linear or branched or acombination thereof. For example, two ASO may be connected head-to-tail(5′-to-3′-linear) (type A) or as in type B, tail-to-tail(3′-to-3′-branched); the ASOs could also be connected head-to-head(5′-to-5′-branched). Similarly, three or more antisense molecules can beconnected (examples C, D, E in FIG. 1B). In an alternative embodiment,the multimer can be in the form of a circular nucleic acid.

B. Targeting Oligonucleotides

In some embodiments, multimeric oligonucleotides provided hereincomprise two or more targeting oligonucleotides linked together by acleavable linker. In some embodiments, each targeting oligonucleotidehas a region complementary to a target region of a genomic targetsequence. In some embodiments, the targeting oligonucleotide is anantisense oligonucleotide (ASO), siRNA (e.g., a single stranded siRNA),miRNA sponge, or anti-microRNA antisense oligonucleotide (AMO). In someembodiments, the targeting oligonucleotide binds specifically to atarget RNA in a cell and brings about degradation of the RNA. In someembodiments, the degradation is mediated by RNAse H. In someembodiments, the degradation is mediated by an RNAi pathway. It shouldbe appreciated that unless otherwise apparent from context “a targetingoligonucleotide” or “the targeting oligonucleotide” as referred toherein, generally means at least one of the targeting oligonucleotidespresent in a multimeric compound. Similarly, it should be appreciatedthat unless otherwise apparent from context “a linker” or “the linker,”as referred to herein, generally means at least one of the linkerspresent in a multimeric compound.

As used herein, the term “genomic target sequence” refers to anucleotide sequence of clinical, therapeutic or research interest in agenome (e.g., a mammalian genome, e.g., a human or mouse genome).Typically, a genomic target sequence is a sequence of a genome thatcomprises a gene coding or regulatory region, or that is present withina gene coding or regulatory region. In some embodiments, a genomictarget sequence is a sequence that encodes at least a portion of a gene.The gene may be an non-coding RNA gene or a protein coding gene. Thenon-coding RNA gene may be a long non-coding RNA gene or an miRNA gene,for example. The product of the gene may be an RNA or protein thatmediates gene expression through an epigenetic mechanism. In otherembodiments, a genomic target sequence is a sequence positioned in aregulatory region of one or more genes, such as a promoter, enhancer,silencer region, locus control region and other functional region of agenome.

In some embodiments, the genomic target sequence is present in the sensestrand of a gene. The sense strand or coding strand is the segment ofdouble stranded DNA running from 5′-3′ that is complementary to theantisense strand or template strand of a gene. The sense strand is thestrand of DNA that has the same sequence as the RNA transcribed from thegene (e.g., mRNA, lncRNA, or miRNA), which takes the antisense strand asits template during transcription.

The “target region” of a genomic target sequence is a sequence ofnucleotides that constitutes a hybridization site of a targetingoligonucleotide. The actual target oligonucleotide may hybridize withthe genomic target itself (e.g., a promoter element) or an nucleic acidencoded by the genomic target sequence or containing the genomic targetsequence (e.g., an lncRNA, miRNA, or mRNA). In some embodiments, thetarget region encodes a site on a transcribed RNA, and hybridization ofa targeting oligonucleotide to the site results in inactivation ordegradation of the transcribed RNA. Accordingly, in some embodiments,the targeting oligonucleotides hybridize to a transcribed RNA encoded bya genomic target sequence and inhibit the function and/or effectdegradation of the transcribed RNA. The RNA may be, for example, a longnon-coding RNA (lncRNA), microRNA, or mRNA.

It should be appreciated that multimeric oligonucleotide compoundsprovided herein may comprise two or more targeting oligonucleotides thatare each complementary to the same or different genomic targetsequences, and thus that may regulate the same or different genes. Insome embodiments, the genomic target sequences is present in the sensestrand of different genes. In some embodiments, the genomic targetsequences is present in the sense strand of the same gene.

In some embodiments, the genomic target sequence of at least onetargeting oligonucleotide is or comprises the sequence of a PRC-2associated region. As used herein, the term “PRC2-associated region”refers to a region of a nucleic acid that comprises or encodes asequence of nucleotides that interact directly or indirectly with acomponent of PRC2. A PRC2-associated region may be present in a RNA(e.g., a long non-coding RNA (lncRNA)) that interacts with a PRC2. APRC2-associated region may be present in a DNA that encodes an RNA thatinteracts with PRC2.

In some embodiments, a PRC2-associated region is a region of an RNA thatcrosslinks to a component of PRC2 in response to in situ ultravioletirradiation of a cell that expresses the RNA, or a region of genomic DNAthat encodes that RNA region. In some embodiments, a PRC2-associatedregion is a region of an RNA that immunoprecipitates with an antibodythat targets a component of PRC2, or a region of genomic DNA thatencodes that RNA region. In some embodiments, a PRC2-associated regionis a region of an RNA that immunoprecipitates with an antibody thatbinds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted aboveare components of PRC2), or a region of genomic DNA that encodes thatRNA region.

In some embodiments, a PRC2-associated region is a region of an RNA thatis protected from nucleases (e.g., RNases) in an RNA-immunoprecipitationassay that employs an antibody that targets a component of PRC2, or aregion of genomic DNA that encodes that protected RNA region. In someembodiments, a PRC2-associated region is a region of an RNA that isprotected from nucleases (e.g., RNases) in an RNA-immunoprecipitationassay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4,or a region of genomic DNA that encodes that protected RNA region.

In some embodiments, a PRC2-associated region is a region of an RNAwithin which occur a relatively high frequency of sequence reads in asequencing reaction of products of an RNA-immunoprecipitation assay thatemploys an antibody that targets a component of PRC2, or a region ofgenomic DNA that encodes that RNA region. In some embodiments, aPRC2-associated region is a region of an RNA within which occur arelatively high frequency of sequence reads in a sequencing reaction ofproducts of an RNA-immunoprecipitation assay that employs an antibodythat binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region ofgenomic DNA that encodes that protected RNA region. In such embodiments,the PRC2-associated region may be referred to as a “peak.”

In some embodiments, a PRC2-associated region comprises a sequence of 40to 60 nucleotides that interact with PRC2 complex. In some embodiments,a PRC2-associated region comprises a sequence of 40 to 60 nucleotidesthat encode an RNA that interacts with PRC2. In some embodiments, aPRC2-associated region comprises a sequence of up to 5 kb in length thatcomprises a sequence (e.g., of 40 to 60 nucleotides) that interacts withPRC2. In some embodiments, a PRC2-associated region comprises a sequenceof up to 5 kb in length within which an RNA is encoded that has asequence (e.g., of 40 to 60 nucleotides) that is known to interact withPRC2. In some embodiments, a PRC2-associated region comprises a sequenceof about 4 kb in length that comprise a sequence (e.g., of 40 to 60nucleotides) that interacts with PRC2. In some embodiments, aPRC2-associated region comprises a sequence of about 4 kb in lengthwithin which an RNA is encoded that includes a sequence (e.g., of 40 to60 nucleotides) that is known to interact with PRC2.

In some embodiments, a PRC2-associated region has a sequence as setforth in SEQ ID NOS: 632,564, 1 to 916,209, or 916,626 to 934,931 ofInternational Patent Appl. Pub. No.: WO/2012/087983, or SEQ ID NOS: 1 to193,049 of International Patent Appl. Pub. No.: WO/2012/065143, each ofwhich is entitled, POLYCOMB-ASSOCIATED NON-CODING RNAS, and the contentsof each of which are incorporated by reference herein in theirentireties.

In some embodiments, the targeting oligonucleotides interfere with thebinding of and function of PRC2 by preventing recruitment of PRC2 to aspecific chromosomal locus through lncRNAs. For example, in someembodiments, administration of multimeric oligonucleotide compoundscomprising targeting oligonucleotides designed to specifically bind aPRC2-associated region of a lncRNA can stably displace not only thelncRNA, but also the PRC2 that binds to the lncRNA, from bindingchromatin. Further, lncRNA can recruit PRC2 in a cis fashion, repressinggene expression at or near the specific chromosomal locus from which thelncRNA was transcribed. Thus, in some embodiments, the compoundsdisclosed herein may be used to inhibit cis mediated gene repression bylncRNAs.

In some embodiments, targeting oligonucleotides may comprise at leastone ribonucleotide, at least one deoxyribonucleotide, and/or at leastone bridged nucleotide. In some embodiments, the oligonucleotide maycomprise a bridged nucleotide, such as a locked nucleic acid (LNA)nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridgednucleic acid (ENA) nucleotide. Examples of such nucleotides aredisclosed herein and known in the art. In some embodiments, theoligonucleotide comprises a nucleotide analog disclosed in one of thefollowing United States patent or patent application Publications: U.S.Pat. No. 7,399,845, U.S. Pat. No. 7,741,457, U.S. Pat. No. 8,022,193,U.S. Pat. No. 7,569,686, U.S. Pat. No. 7,335,765, U.S. Pat. No.7,314,923, U.S. Pat. No. 7,335,765, and U.S. Pat. No. 7,816,333, US20110009471, the entire contents of each of which are incorporatedherein by reference for all purposes. The targeting oligonucleotide mayhave one or more 2′ O-methyl nucleotides. The oligonucleotide mayconsist entirely of 2′ O-methyl nucleotides.

The targeting oligonucleotide may contain one or more nucleotideanalogues. For example, the targeting oligonucleotide may have at leastone nucleotide analogue that results in an increase in T_(m) of theoligonucleotide in a range of 1° C., 2° C., 3° C., 4° C., or 5° C.compared with an oligonucleotide that does not have the at least onenucleotide analogue. The targeting oligonucleotide may have a pluralityof nucleotide analogues that results in a total increase in T_(m) of theoligonucleotide in a range of 2° C., 3° C., 4° C., 5° C., 6° C., 7° C.,8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C.,45° C. or more compared with an oligonucleotide that does not have thenucleotide analogue.

In some embodiments, the targeting oligonucleotide may be of up to 50nucleotides in length or up to 100 nucleotides in length, in which 2 to10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to30, 2 to 40, 2 to 45, 2 to 75, 2 to 95, or more nucleotides of theoligonucleotide are nucleotide analogues. The oligonucleotide may be of8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of theoligonucleotide are nucleotide analogues. The oligonucleotide may be of8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides ofthe oligonucleotide are nucleotide analogues. Optionally, theoligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 nucleotides modified.

The targeting oligonucleotide may consist entirely of bridgednucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides).The oligonucleotide may comprise alternating deoxyribonucleotides and2′-fluoro-deoxyribonucleotides. The oligonucleotide may comprisealternating deoxyribonucleotides and 2′-O-methyl nucleotides. Theoligonucleotide may comprise alternating deoxyribonucleotides and ENAnucleotide analogues. The oligonucleotide may comprise alternatingdeoxyribonucleotides and LNA nucleotides. The oligonucleotide maycomprise alternating LNA nucleotides and 2′-O-methyl nucleotides. Theoligonucleotide may have a 5′ nucleotide that is a bridged nucleotide(e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). Theoligonucleotide may have a 5′ nucleotide that is a deoxyribonucleotide.

The targeting oligonucleotide may comprise deoxyribonucleotides flankedby at least one bridged nucleotide (e.g., a LNA nucleotide, cEtnucleotide, ENA nucleotide) on each of the 5′ and 3′ ends of thedeoxyribonucleotides. The oligonucleotide may comprisedeoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridgednucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) oneach of the 5′ and 3′ ends of the deoxyribonucleotides. The 3′ positionof the oligonucleotide may have a 3′ hydroxyl group. The 3′ position ofthe oligonucleotide may have a 3′ thiophosphate.

The targeting oligonucleotide may be conjugated with a label. Forexample, the oligonucleotide may be conjugated with a biotin moiety,cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers,peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR ordynamic polyconjugates and variants thereof at its 5′ or 3′ end.

The targeting oligonucleotide may comprise one or more modificationscomprising: a modified sugar moiety, and/or a modified internucleosidelinkage, and/or a modified nucleotide and/or combinations thereof. It isnot necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the modificationsdescribed herein may be incorporated in a single oligonucleotide or evenat within a single nucleoside within an oligonucleotide.

In some embodiments, the targeting oligonucleotides are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimerictargeting oligonucleotides of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the targeting oligonucleotide comprises at leastone nucleotide modified at the 2′ position of the sugar, most preferablya 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Inother preferred embodiments, RNA modifications include 2′-fluoro,2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues or an inverted base at the 3′ end of the RNA. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than a native oligodeoxynucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides, in some experimental or therapeutics contexts.Specific examples of modified oligonucleotides include those comprisingmodified backbones, for example, phosphorothioates, phosphotriesters,methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkagesor short chain heteroatomic or heterocyclic intersugar linkages. Mostpreferred are oligonucleotides with phosphorothioate backbones and thosewith heteroatom backbones, particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂(known as a methylene(methylimino) or MMI backbone, CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholinobackbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506);peptide nucleic acid (PNA) backbone (wherein the phosphodiester backboneof the oligonucleotide is replaced with a polyamide backbone, thenucleotides being bound directly or indirectly to the aza nitrogen atomsof the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991. In some embodiments, the morpholino-basedoligomeric compound is a phosphorodiamidate morpholino oligomer (PMO)(e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001;and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures ofwhich are incorporated herein by reference in their entireties).

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

Modified oligonucleotides are also known that include oligonucleotidesthat are based on or constructed from arabinonucleotide or modifiedarabinonucleotide residues. Arabinonucleosides are stereoisomers ofribonucleosides, differing only in the configuration at the 2′-positionof the sugar ring. In some embodiments, a 2′-arabino modification is2′-F arabino. In some embodiments, the modified oligonucleotide is2′-fluoro-D-arabinonucleic acid (FANA) (as described in, for example,Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med.Chem. Lett., 12:2651-2654, 2002; the disclosures of which areincorporated herein by reference in their entireties). Similarmodifications can also be made at other positions on the sugar,particularly the 3′ position of the sugar on a 3′ terminal nucleoside orin 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide.

PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA)oligomers and their analogues for improved sequence specific inhibitionof gene expression via association to complementary messenger RNA.

Other preferred modifications include ethylene-bridged nucleic acids(ENAs) (e.g., International Patent Publication No. WO 2005/042777,Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al.,Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther.,8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf),49:171-172, 2005; the disclosures of which are incorporated herein byreference in their entireties). Preferred ENAs include, but are notlimited to, 2′-O,4′-C-ethylene-bridged nucleic acids.

Examples of LNAs are described in WO/2008/043753 and include compoundsof the following general formula.

in which X and Y are independently selected among the groups —O—, —S—,—N(H)—, N(R)—, —CH₂— or —CH— (if part of a double bond), —CH₂—O—,—CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if part of adouble bond), —CH═CH—, where R is selected from hydrogen and C₁₋₄-alkyl;Z and Z* are independently selected among an internucleoside linkage, aterminal group or a protecting group; B constitutes a natural ornon-natural nucleotide base moiety; and the asymmetric groups may befound in either orientation.

Preferably, the LNA used in the oligonucleotides described hereincomprises at least one LNA unit according any of the formulas

in which Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleoside linkage, a terminal group or aprotecting group; B constitutes a natural or non-natural nucleotide basemoiety, and RH is selected from hydrogen and C₁₋₄-alkyl.

Preferably, the Locked Nucleic Acid (LNA) used in the oligonucleotidesdescribed herein comprises at least one nucleotide comprises a LockedNucleic Acid (LNA) unit according any of the formulas shown in Scheme 2of PCT/DK2006/000512.

Preferably, the LNA used in the oligomer of the invention comprisesinternucleoside linkages selected from -0-P(O)₂—O—, —O—P(O,S)—O—,-0-O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, -0-P(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, -0-PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected from hydrogen and C₁₋₄-alkyl.

Specifically preferred LNA units are shown in scheme 2:

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from S or —CH₂—S—.Thio-LNA can be in both beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from —N(H)—, N(R)—,CH₂—N(H)—, and —CH₂—N(R)— where R is selected from hydrogen andC₁₋₄-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above represents —O— or —CH₂—O—.Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is —CH₂—O— (where the oxygen atom of —CH₂—O— isattached to the 2′-position relative to the base B).

LNAs are described in additional detail herein.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃,OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-0-CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)] (Martin et al, HeIv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy(2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Targeting oligonucleotides can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, aswell as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,5-propynyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,2-chloro-6-aminopurine and 2,6-diaminopurine or other diaminopurines.See, e.g., Kornberg, “DNA Replication,” W. H. Freeman & Co., SanFrancisco, 1980, pp 75-77; and Gebeyehu, G., et al. Nucl. Acids Res.,15:4513 (1987)). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, in Crooke, andLebleu, eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and may be used as base substitutions. Itshould be appreciated that one or more modified bases may be present ina region of complementarity of a targeting oligonucleotide.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the modificationsdescribed herein may be incorporated in a single oligonucleotide or evenat within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science, 1991, 254, 1497-1500.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in “The Concise Encyclopedia of PolymerScience And Engineering”, pages 858-859, Kroschwitz, ed. John Wiley &Sons, 1990; those disclosed by Englisch et al., Angewandle Chemie,International Edition, 1991, 30, page 613, and those disclosed bySanghvi, Chapter 15, Antisense Research and Applications,” pages289-302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, etal., eds, “Antisense Research and Applications,” CRC Press, Boca Raton,1993, pp. 276-278) and are presently preferred base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications. Modified nucleobases are described in U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617;5,750,692, and 5,681,941, each of which is herein incorporated byreference.

In some embodiments, the targeting oligonucleotides are chemicallylinked to one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Forexample, one or more targeting oligonucleotides, of the same ordifferent types, can be conjugated to targeting moieties with enhancedspecificity for a cell type or tissue type. Such moieties include, butare not limited to, lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al,Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996,277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and5,688,941, each of which is herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

In some embodiments, targeting oligonucleotide modification includemodification of the 5′ or 3′ end of the oligonucleotide. In someembodiments, the 3′ end of the oligonucleotide comprises a hydroxylgroup or a thiophosphate. It should be appreciated that additionalmolecules (e.g. a biotin moiety or a fluorophor) can be conjugated tothe 5′ or 3′ end of the targeting oligonucleotide. In some embodiments,the targeting oligonucleotide comprises a biotin moiety conjugated tothe 5′ nucleotide.

In some embodiments, the targeting oligonucleotide comprises lockednucleic acids (LNA), ENA modified nucleotides, 2′-O-methyl nucleotides,or 2′-fluoro-deoxyribonucleotides. In some embodiments, the targetingoligonucleotide comprises alternating deoxyribonucleotides and2′-fluoro-deoxyribonucleotides. In some embodiments, the targetingoligonucleotide comprises alternating deoxyribonucleotides and2′-O-methyl nucleotides. In some embodiments, the targetingoligonucleotide comprises alternating deoxyribonucleotides and ENAmodified nucleotides. In some embodiments, the targeting oligonucleotidecomprises alternating deoxyribonucleotides and locked nucleic acidnucleotides. In some embodiments, the targeting oligonucleotidecomprises alternating locked nucleic acid nucleotides and 2′-O-methylnucleotides.

In some embodiments, the 5′ nucleotide of the oligonucleotide is adeoxyribonucleotide. In some embodiments, the 5′ nucleotide of theoligonucleotide is a locked nucleic acid nucleotide. In someembodiments, the nucleotides of the oligonucleotide comprisedeoxyribonucleotides flanked by at least one locked nucleic acidnucleotide on each of the 5′ and 3′ ends of the deoxyribonucleotides. Insome embodiments, the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group or a 3′ thiophosphate.

In some embodiments, the targeting oligonucleotide comprisesphosphorothioate internucleotide linkages. In some embodiments, thetargeting oligonucleotide comprises phosphorothioate internucleotidelinkages between at least two nucleotides. In some embodiments, thetargeting oligonucleotide comprises phosphorothioate internucleotidelinkages between all nucleotides.

It should be appreciated that the targeting oligonucleotide can have anycombination of modifications as described herein.

It should also be appreciated that oligonucleotide based linkers mayalso include any of the modifications disclosed herein.

C. Antisense-Based Targeting Oligonucleotides

In illustrative embodiments, the targeting oligonucleotides aretargeting oligonucleotides that contain locked nucleic acid 3-8-3gapmers which have a phosphorothioate backbone. However, in general, thechemistry of the oligonucleotide is not limited to LNA(2′-O,4′-C-methylene-bridged nucleic acids described, e.g., in PCTpatent application WO 98/39352), LNA gapmers, or the phosphorothioatebackbone, and can be expected to work with any chemistry for which thetarget knock-down using a monomeric ASO is effective. Such chemistriesinclude, for instance, 2′-O,4′-C-ethylene-bridged nucleic acids (ENA;European patent No. EP 1152009), hexitol nucleic acids (HNA; WO 93/25565and WO 97/30064), fluoro-HNA, 2′-deoxy-2′-fluoro-13-D-arabino nucleicacids (FANA; EP 1088066), 2′-modified analogs such as 2′-O-methyl(2′-OMe) and 2′-O-(2-methoxyethyl) (MOE) modified nucleic acids, CeNA(EP 1210347 and EP 1244667) as well as phosphate-modified analogs suchas phosphoroamidate, morpholinos, base-modified analogs, such asG-clamps (WO 99/24452) and 5-alkynyl-pyrimidines. Examples of LNA othergapmers are described in PCT patent applications published as WO01/25248, WO 01/48190, WO 2003/085110, WO 2004/046160, WO 2008/113832,WO 2005/023825 and WO 2007/14651; examples of FANA/DNA/FANA gapmers aredescribed in EP 1315807; examples of 2′-OMe/FANA/2′-OMe gapmers aredescribed in U.S. Pat. No. 6,673,611.

The backbone may be stabilized by other modifications, for example,methylphosphonate or other chemistries. The antisense oligonucleotidesof this invention can work via an RNase H mechanism, but can also workby steric blocking only, which also includes transcriptional genesilencing and transcriptional gene activation (see, e.g., Hawkins etal., 2009, Nucl. Acids Res., 37(9):2984-2995 and Schwartz et al., 2008,Nature Struct. Mol. Biol., 15:842-848). The dimer/multimer approach canalso be combined with any modification which increases the delivery intocells, including lipophilic modifications, conjugates to cell surfacereceptors or ligands (e.g., folate), aptamers, etc. For example, toexploit the RNAse H mechanism, DNA:mRNA or gapmer:RNA duplexes need tobe formed to permit RNAse to bind to the substrate. However, in the caseof steric blocking, RNA:RNA, RNA:2′-O-methyl-RNA, RNA:PNA or RNA:LNAduplexes (without a DNA gap) may be used. Thus, the ASO chemistry may beadjusted based on the intended use. Any chemistry suitable for theantisense oligonucleotides should be applicable to the dimer/multimerapproach of the invention (for the state-of-the-art chemistries, see,e.g., Bennett and Swaize, 2009, Ann. Rev. Pharmacol. Toxicol.,50:259-293; Yokota at al., 2010, Arch. Neurol., 66:32-38; Aboul-Fadl,2005, Curr. Med. Chem., 12:2193-2214; Kurreck, 2003, Eur. J. Biochem.,270:1628-1644).

In some illustrative embodiments, the targeting oligonucleotides are14-nucleotide long, but could be generally longer or shorter. Forexample, the targeting oligonucleotide could be 8-50-nucleotide long, or10-40, 10-25, 8-20, 10-25, 12-25, 12-20, 12-16, 12-15, 12-14, 12-13,13-16, 13-15, or 13-14 nucleotides long. In some embodiments, targetingoligonucleotides are so-called tiny LNAs, containing as few as 8 orfewer nucleotides (see, e.g., Obad et al. (2011) Nature Genetics,43:371).

Further, in some embodiments, a targeting oligonucleotide (e.g., ASO)comprises at least 7 contiguous nucleotides complementary to the targetsequence. In further embodiments, targeting oligonucleotide (e.g., ASO)comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, or 40 contiguous nucleotides complementary to atarget sequence. Due to specificities and isoform variations, ASO mayadditionally comprise 1, 2, 3 or more non-complementary nucleotides,either within the contiguous sequences or flanking them. In someembodiments, at least one or all of the targeting oligonucleotides aregapmers. In other embodiments, the targeting oligonucleotides (e.g.,ASOs) are X—N—Y gapmers, wherein at X or Y contains 0, 1, 2, 3, 4, 5 ormore modified nucleotides, e.g., LNA, ENA, FANA, G-clamp, and N is 3, 4,5, 6, 7, 8, 9, or 10 deoxynucleotides with non-modified sugars. Forexample, ASO can be a 3-8-3, 2-10-2, 3-9-2, 2-9-3, 2-8-2, 3-7-2, 2-7-3gapmer or another type of gapmer or mixmer.

D. Linkers

The term “linker” generally refers to a chemical moiety that is capableof covalently linking two or more targeting oligonucleotides, in whichat least one bond comprised within the linker is capable of beingcleaved (e.g., in a biological context, such as in a mammalian extract,such as an endosomal extract), such that at least two targetingoligonucleotides are no longer covalently linked to one another afterbond cleavage. It will be appreciated that a provided linker may includea region that is non-cleavable, as long as the linker also comprises atleast one bond that is cleavable.

In some embodiments, the linker comprises a polypeptide that is moresusceptible to cleavage by an endopeptidase in the mammalian extractthan the targeting oligonucleotides. The endopeptidase may be a trypsin,chymotrypsin, elastase, thermolysin, pepsin, or endopeptidase V8. Theendopeptidase may be a cathepsin B, cathepsin D, cathepsin L, cathepsinC, papain, cathepsin S or endosomal acidic insulinase. For example, thelinker comprise a peptide having an amino acid sequence selected from:ALAL (SEQ ID NO: 125), APISFFELG (SEQ ID NO: 126), FL, GFN, R/KXX,GRWHTVGLRWE (SEQ ID NO: 127), YL, GF, and FF, in which X is any aminoacid.

In some embodiments, the linker comprises the formula—(CH₂)_(n)S—S(CH₂)_(m)—, wherein n and m are independently integers from0 to 10.

For example, the linker of a multimeric oligonucleotide may comprise anoligonucleotide that is more susceptible to cleavage by an endonucleasein the mammalian extract than the targeting oligonucleotides. The linkermay have a nucleotide sequence comprising from 1 to 10 thymidines oruridines. The linker may have a nucleotide sequence comprisingdeoxyribonucleotides linked through phosphodiester intemucleotidelinkages. The linker may have a nucleotide sequence comprising from 1 to10 thymidines linked through phosphodiester intemucleotide linkages. Thelinker may have a nucleotide sequence comprising from 1 to 10 uridineslinked through phosphorothioate intemucleotide linkages. The linker mayhave the formula:

in which Z is an oligonucleotide. Z may have a nucleotide sequencecomprising from 1 to 10 thymidines or uridines.

In some embodiments, the linker does not comprise an oligonucleotidehaving a self-complementary nucleotide sequence. In some embodiments,the linker does not comprise an oligonucleotide having a nucleotidesequence that is complementary to a region of the genomic targetsequence that is contiguous with two flanking target regions. In someembodiments, the linker does not comprise an oligonucleotide having aself-complementary nucleotide sequence and does not comprise anoligonucleotide having a nucleotide sequence that is complementary to aregion of the genomic target sequence that is contiguous with twoflanking target regions of the particular linker. In some embodiments,the at least one L is a linker that does not comprise an oligonucleotidehaving an abasic site.

In other embodiments, multimeric oligonucleotide compounds are providedthat comprise at least two targeting oligonucleotides each of which islinked to one or two other targeting oligonucleotides through a linker.In some embodiments, at least one linker is 2-fold, 3-fold, 4-fold,5-fold, 10-fold or more sensitive to enzymatic cleavage in the presenceof a mammalian extract than at least two targeting oligonucleotides. Itshould be appreciated that different linkers can be designed to becleaved at different rates and/or by different enzymes in compoundscomprising two or more linkers. Similarly different linkers can bedesigned to be sensitive to cleavage in different tissues, cells orsubcellular compartments in compounds comprising two or more linkers.This can advantageously permit compounds to have targetingoligonucleotides that are released from compounds at different rates, bydifferent enzymes, or in different tissues, cells or subcellularcompartments thereby controlling release of the monomericoligonucleotides to a desired in vivo location or at a desired timefollowing administration.

In some embodiments, the invention also provides ASO multimerscomprising targeting oligonucleotides having nuclease-resistant backbone(e.g., phosphorothioate), wherein the targeting oligonucleotides arelinked to each other by one or more cleavable linkers.

In certain embodiments, linkers are stable in plasma, blood or serumwhich are richer in exonucleases, and less stable in the intracellularenvironments which are relatively rich in endonucleases. Theintracellular stability of linkers can be assessed in vitro or in vivoas described in the Examples. In some embodiments, a linker isconsidered “non-cleavable” if the linker's half-life is at least 24, or28, 32, 36, 48, 72, 96 hours or longer under the conditions describedhere, such as in liver homogenates. Conversely, in some embodiments, alinker is considered “cleavable” if the half-life of the linker is atmost 10, or 8, 6, 5 hours or shorter.

In some embodiments, the linker is a nuclease-cleavable oligonucleotidelinker. In some embodiments, the nuclease-cleavable linker contains oneor more phosphodiester bonds in the oligonucleotide backbone. Forexample, the linker may contain a single phosphodiester bridge or 2, 3,4, 5, 6, 7 or more phosphodiester linkages, for example as a string of1-10 deoxynucleotides, e.g., dT, or ribonucleotides, e.g., rU, in thecase of RNA linkers. In the case of dT or other DNA nucleotides dN inthe linker, in certain embodiments the cleavable linker contains one ormore phosphodiester linkages. In other embodiments, in the case of rU orother RNA nucleotides rN, the cleavable linker may consist ofphosphorothioate linkages only. In contrast to phosphorothioate-linkeddeoxynucleotides, which are only cleaved slowly by nucleases (thustermed “noncleavable”), phosphorothioate-linked rU undergoes relativelyrapid cleavage by ribonucleases and therefore is considered cleavableherein. It is also possible to combine dN and rN into the linker region,which are connected by phosphodiester or phosphorothioate linkages. Inother embodiments, the linker can also contain chemically modifiednucleotides, which are still cleavable by nucleases, such as, e.g.,2′-O-modified analogs. In particular, 2′-O-methyl or 2′-fluoronucleotides can be combined with each other or with dN or rNnucleotides. Generally, in the case of nucleotide linkers, the linker isa part of the multimer that is usually not complementary to a target,although it could be. This is because the linker is generally cleavedprior to targeting oligonucleotides action on the target, and therefore,the linker identity with respect to a target is inconsequential.Accordingly, in some embodiments, a linker is an (oligo)nucleotidelinker that is not complementary to any of the targets against which thetargeting oligonucleotides are designed.

In some embodiments, the cleavable linker is oligonucleotide linker thatcontains a continuous stretch of deliberately introduced Rpphosphorothioate stereoisomers (e.g., 4, 5, 6, 7 or longer stretches).The Rp stereoisoform, unlike Sp isoform, is known to be susceptible tonuclease cleavage (Krieg et al., 2003, Oligonucleotides, 13:491-499).Such a linker would not include a racemic mix of PS linkagesoligonucleotides since the mixed linkages are relatively stable and arenot likely to contain long stretches of the Rp stereoisomers, andtherefore, considered “non-cleavable” herein. Thus, in some embodiments,a linker comprises a stretch of 4, 5, 6, 7 or more phosphorothioatednucleotides, wherein the stretch does not contain a substantial amountor any of the Sp stereoisoform. The amount could be consideredsubstantial if it exceeds 10% on per-mole basis.

In some embodiments, the linker is a non-nucleotide linker, for example,a single phosphodiester bridge. Another example of such cleavablelinkers is a chemical group comprising a disulfide bond, for example,—(CH₂)_(n)S—S(CH₂)_(m)—, wherein n and m are integers from 0 to 10. Inillustrative embodiments, n=m=6. Additional example of non-nucleotidelinkers are described below.

The cleavable linkers may be present in other linear or branchedmultimers. For example in some branched embodiments, the cleavablelinker comprises a “doubler,” “trebler,” or another branching chemicalgroup with multiple “arms” that link phosphodiester linked nucleotides,as for example, illustrated in FIGS. 1C and 1D and Formulas IV, V, andVIII. In some linear embodiments, cleavable linkers can be incorporatedas shown in Formulas I and II.

The linker can be designed so as to undergo a chemical or enzymaticcleavage reaction. Chemical reactions involve, for example, cleavage inacidic environment (e.g., endosomes), reductive cleavage (e.g.,cytosolic cleavage) or oxidative cleavage (e.g., in liver microsomes).The cleavage reaction can also be initiated by a rearrangement reaction.Enzymatic reactions can include reactions mediated by nucleases,peptidases, proteases, phosphatases, oxidases, reductases, etc. Forexample, a linker can be pH-sensitive, cathepsin-sensitive, orpredominantly cleaved in endosomes and/or cytosol.

In some embodiments, the linker comprises a peptide. In certainembodiments, the linker comprises a peptide which includes a sequencethat is cleavable by an endopeptidase. In addition to the cleavablepeptide sequence, the linker may comprise additional amino acid residuesand/or non-peptide chemical moieties, such as an alkyl chain. In certainembodiments, the linker comprises Ala-Leu-Ala-Leu (SEQ ID NO.: 125),which is a substrate for cathepsin B. See, for example, themaleimidocaproyl-Arg-Arg-Ala-Leu-Ala-Leu (SEQ ID NO.: 136) linkersdescribed in Schmid et al, Bioconjugate Chem 2007, 18, 702-716. Incertain embodiments, a cathepsin B-cleavable linker is cleaved in tumorcells. In certain embodiments, the linker comprisesAla-Pro-Ile-Ser-Phe-Phe-Glu-Leu-Gly (SEQ ID NO.: 126), which is asubstrate for cathepsins D, L, and B (see, for example, Fischer et al,Chembiochem 2006, 7, 1428-1434). In certain embodiments, acathepsin-cleavable linker is cleaved in HeLA cells. In someembodiments, the linker comprises Phe-Lys, which is a substrate forcathepsin B. For example, in certain embodiments, the linker comprisesPhe-Lys-p-aminobenzoic acid (PABA). See, e.g., themaleimidocaproyl-Phe-Lys-PABA linker described in Walker et al, Bioorg.Med. Chem. Lett. 2002, 12, 217-219. In certain embodiments, the linkercomprises Gly-Phe-2-naphthylamide, which is a substrate for cathepsin C(see, for example, Berg et al. Biochem. J. 1994, 300, 229-235). Incertain embodiments, a cathepsin C-cleavable linker is cleaved inhepatocytes, In some embodiments, the linker comprises a cathepsin Scleavage site. For example, in some embodiments, the linker comprisesGly-Arg-Trp-His-Thr-Val-Gly-Leu-Arg-Trp-Glu (SEQ ID NO.: 127),Gly-Arg-Trp-Pro-Pro-Met-Gly-Leu-Pro-Trp-Glu (SEQ ID NO.: 137), orGly-Arg-Trp-His-Pro-Met-Gly-Ala-Pro-Trp-Glu (SEQ ID NO.: 138), forexample, as described in Lutzner et al, J. Biol. Chem. 2008, 283,36185-36194. In certain embodiments, a cathepsin S-cleavable linker iscleaved in antigen presenting cells. In some embodiments, the linkercomprises a papain cleavage site. Papain typically cleaves a peptidehaving the sequence -R/K-X-X (see Chapman et al, Annu. Rev. Physiol1997, 59, 63-88). In certain embodiments, a papain-cleavable linker iscleaved in endosomes. In some embodiments, the linker comprises anendosomal acidic insulinase cleavage site. For example, in someembodiments, the linker comprises Tyr-Leu, Gly-Phe, or Phe-Phe (see,e.g., Authier et al, FEBS Lett. 1996, 389, 55-60). In certainembodiments, an endosomal acidic insulinase-cleavable linker is cleavedin hepatic cells.

In some embodiments, the linker is pH sensitive. In certain embodiments,the linker comprises a low pH-labile bond. As used herein, a low-pHlabile bond is a bond that is selectively broken under acidic conditions(pH<7). Such bonds may also be termed endosomally labile bonds, becausecell endosomes and lysosomes have a pH less than 7. For example, incertain embodiments, the linker comprises an amine, an imine, an ester,a benzoic imine, an amino ester, a diortho ester, a polyphosphoester, apolyphosphazene, an acetal, a vinyl ether, a hydrazone, anazidomethyl-methylmaleic anhydride, a thiopropionate, a maskedendosomolytic agent or a citraconyl group.

In certain embodiments, the linker comprises a low pH-labile bondselected from the following: ketals that are labile in acidicenvironments (e.g., pH less than 7, greater than about 4) to form a dioland a ketone; acetals that are labile in acidic environments (e.g., pHless than 7, greater than about 4) to form a diol and an aldehyde;imines or iminiums that are labile in acidic environments (e.g., pH lessthan 7, greater than about 4) to form an amine and an aldehyde or aketone; silicon-oxygen-carbon linkages that are labile under acidiccondition; silicon-nitrogne (silazane) linkages; silicon-carbon linkages(e.g., arylsilanes, vinylsilanes, and allylsilanes); maleamates (amidebonds synthesized from maleic anhydride derivatives and amines); orthoesters; hydrazones; activated carboxylic acid derivatives (e.g., esters,amides) designed to undergo acid catalyzed hydrolysis); or vinyl ethers.Further examples may be found in International Patent Appln. Pub. No. WO2008/022309, entitled POLYCONJUGATES FOR IN VIVO DELIVERY OFPOLYNUCLEOTIDES, the contents of which are incorporated herein byreference.

Organosilanes (e.g., silyl ethers, silyl enol ethers) are used as oxygenprotecting groups in organic synthesis. Silicon-oxygen-carbon linkagesare susceptible to hydrolysis under acidic conditions to form silanolsand an alcohol (or enol). The substitution on both the silicon atom andthe alcohol carbon can affect the rate of hydrolysis due to steric andelectronic effects. This allows for the possibility of tuning the rateof hydrolysis of the silicon-oxygen-carbon linkage by changing thesubstitution on either the organosilane, the alcohol, or both theorganosilane and alcohol. In addition, charged or reactive groups, suchas amines or carboxylate, may be attached to the silicon atom, whichconfers the labile compound with charge and/or reactivity.

Hydrolysis of a silazane leads to the formation of a silanol and anamine. Silazanes are inherently more susceptible to hydrolysis than isthe silicon-oxygen-carbon linkage, however, the rate of hydrolysis isincreased under acidic conditions. The substitution on both the siliconatom and the amine can affect the rate of hydrolysis due to steric andelectronic effects. This allows for the possibility of tuning the rateof hydrolysis of the silazane by changing the substitution on either thesilicon or the amine.

Another example of a pH labile bond is an acid labile enol ether bond.The rate at which this labile bond is cleaved depends on the structuresof the carbonyl compound formed and the alcohol released. For exampleanalogs of ethyl isopropenyl ether, which may be synthesized fromβ-haloethers, generally have shorter half lives than analogs of ethylcyclohexenyl ether, which may be synthesized from phenol ethers

Reaction of an anhydride with an amine forms an amide and an acid.Typically, the reverse reaction (formation of an anhydride and amine) isvery slow and energetically unfavorable. However, if the anhydride is acyclic anhydride, reaction with an amine yields a molecule in which theamide and the acid are in the same molecule, an amide acid. The presenceof both reactive groups (the amide and the carboxylic acid) in the samemolecule accelerates the reverse reaction. In certain embodiments, thelinker comprises maleamic acid. Cleavage of the amide acid to form anamine and an anhydride is pH-dependent, and is greatly accelerated atacidic pH. This pH-dependent reactivity can be exploited to formreversible pH-sensitive bonds and linkers. Cis-aconitic acid has beenused as such a pH-sensitive linker molecule. The γ-carboxylate is firstcoupled to a molecule. In a second step, either the at or 3 carboxylateis coupled to a second molecule to form a pH-sensitive coupling of thetwo molecules.

In some embodiments, the linker comprises a benzoic imine as a low-pHlabile bond. See, for example, the conjugates described in Zhu et al,Langmuir 2012, 28, 11988-96; Ding et al, Bioconjug. Chem. 2009, 20,1163-70.

In some embodiments, the linker comprises a low pH-labile hydrazonebond. Such acid-labile bonds have been extensively used in the field ofconjugates, e.g., antibody-drug conjugates. See, for example, Zhou etal, Biomacromolecules 2011, 12, 1460-7; Yuan et al, Acta Biomater. 2008,4, 1024-37; Zhang et al, Acta Biomater. 2007, 6, 838-50; Yang et al, J.Pharmacol. Exp. Ther. 2007, 321, 462-8; Reddy et al, Cancer Chemother.Pharmacol. 2006, 58, 229-36; Doronina et al, Nature Biotechnol. 2003,21, 778-84. In some embodiments, the linker comprises a low pH-labilevinyl ether. See, for example, Shin et al, J. Control. Release 2003, 91,187-200. In some embodiments, the linker comprises a low pH-labilephosphoamine bond. In some embodiments, the linker comprises a lowpH-labile traceless click linker. For example, in certain embodiments,the linker comprises azidomethyl-methylmaleic anhydride (see Maier etal, J. Am. Chem. Soc. 2012 134, 10169-73. In some embodiments, thelinker comprises a low pH-labile 4-hydrazinosulfonyl benzoic acidlinker. See, for example, Kaminskas et al, Mol. Pharm. 2012 9, 422-32;Kaminskas et al, J. Control. Release 2011, 152, 241-8. In someembodiments, the linker comprises a low pH-labile para-phenylpropionicacid linker (see, e.g., Indira Chandran et al, Cancer Lett. 2012 316,151-6). In some embodiments, the linker comprises a low pH-labileβ-thiopropionate linker (see, e.g., Dan et al, Langmuir 2011, 27,612-7). In some embodiments, the linker comprises a low pH-labile ester(see, for example, Zhu et al, Bioconjug. Chem. 2010, 21, 2119-27). Insome embodiments, the linker comprises a low pH-labile ketal (see, e.g.,Abraham et al, J. Biomater. Sci. Polym. Ed. 2011, 22, 1001-22) or acetal(see, e.g., Liu et al, J. Am. Chem. Soc. 2010, 132, 1500). In someembodiments, the linker comprises a low pH-labile4-(4′-acetylphenoxy)butanoic acid linker (see, e.g., DiJoseph et al,Blood 2004, 103, 1807-14). In some embodiments, the linker comprises alow pH-labile cis-aconityl linker (see, e.g., Haas et al, J. Drug Target2002, 10, 81-9; Ahmad et al, Anticancer Res. 1990, 10, 837-43; Dillmanet al, Cancer Res. 1988, 48, 6097-102). In some embodiments, the linkercomprises a low pH-labile diortho ester (see, e.g, Guo et al, Bioconjug.Chem. 2001, 12, 291-300).

In some embodiments, the linker comprises a masked endosomolytic agent.Endosomolytic polymers are polymers that, in response to a change in pH,are able to cause disruption or lysis of an endosome or provide forescape of a normally membrane-impermeable compound, such as apolynucleotide or protein, from a cellular internal membrane-enclosedvesicle, such as an endosome or lysosome. A subset of endosomolyticcompounds is fusogenic compounds, including fusogenic peptides.Fusogenic peptides can facilitate endosomal release of agents such asoligomeric compounds to the cytoplasm. See, for example, US PatentApplication Publication Nos. 20040198687, 20080281041, 20080152661, and20090023890, which are incorporated herein by reference.

The linker can also be designed to undergo an organ/tissue-specificcleavage. For example, for certain targets, which are expressed inmultiple tissues, only the knock-down in liver may be desirable, asknock-down in other organs may lead to undesired side effects. Thus,linkers susceptible to liver-specific enzymes, such as pyrrolase (TPO)and glucose-6-phosphatase (G-6-Pase), can be engineered, so as to limitthe antisense effect to the liver mainly. Alternatively, linkers notsusceptible to liver enzymes but susceptible to kidney-specific enzymes,such as gamma-glutamyltranspeptidase, can be engineered, so that theantisense effect is limited to the kidneys mainly. Analogously,intestine-specific peptidases cleaving Phe-Ala and Leu-Ala could beconsidered for orally administered multimeric targetingoligonucleotides. Similarly, by placing an enzyme recognition site intothe linker, which is recognized by an enzyme over-expressed in tumors,such as plasmin (e.g., PHEA-D-Val-Leu-Lys recognition site),tumor-specific knock-down should be feasible. By selecting the rightenzyme recognition site in the linker, specific cleavage and knock-downshould be achievable in many organs. In addition, the linker can alsocontain a targeting signal, such as N-acetyl galactosamine for livertargeting, or folate, vitamin A or RGD-peptide in the case of tumor oractivated macrophage targeting. Accordingly, in some embodiments, thecleavable linker is organ- or tissue-specific, for example,liver-specific, kidney-specific, intestine-specific, etc.

The targeting oligonucleotides can be linked through any part of theindividual targeting oligonucleotide, e.g., via the phosphate, the sugar(e.g., ribose, deoxyribose), or the nucleobase. In certain embodiments,when linking two oligonucleotides together, the linker can be attachede.g. to the 5′-end of the first oligonucleotide and the 3′-end of thesecond nucleotide, to the 5′-end of the first oligonucleotide and the5′end of the second nucleotide, to the 3′-end of the firstoligonucleotide and the 3′-end of the second nucleotide. In otherembodiments, when linking two oligonucleotides together, the linker canattach internal residues of each oligonucleotides, e.g., via a modifiednucleobase. One of ordinary skill in the art will understand that manysuch permutations are available for multimers.

The linkers described herein can also be used to attach other moietiesto an oligonucleotide. Such moieties include lipophilic moieties,targeting moieties (e.g., a ligand of a cell surface receptor), and tags(e.g., a fluorescent moiety for imaging or an affinity tag such asbiotin).

In certain embodiments, the linker is attached to an oligonucleotide viaclick chemistry (for a review of using click chemistry with DNA, seeEl-Sagheer et al, Chem. Soc. Rev. 2010, 39, 1388-1405). The term “clickchemistry” is used to describe any facile reaction that occurs in highyields, under mild conditions, and in the presence of diverse functionalgroups, but it is most commonly used to refer to a [3+2] azide-alkynecycloaddition reaction. Such reactions are generally catalyzed by Cu^(I)and proceed in the presence of functional groups typically encounteredin biological molecules. In some embodiments, an unnatural base isintroduced into the oligonucleotide, wherein the base is modified tocomprise an alkyne or azide. See below for exemplary base modifications:

-   -   wherein R′ is, for example, hydrogen, a suitable protecting        group or coupling moiety (e.g., 4,4′-dimethoxytrityl (DMT), or a        phosphoramidite group), a triphosphate, or R′ denotes the point        of connection to the rest of an oligonucleotide.

In some embodiments, an oligonucleotide is modified such that the ribosemoiety comprises an alkyne or azide for coupling the linker. Forexample:

In some embodiments, an oligonucleotide is modified on the 5′ or 3′ endwith an alkyne or azide for coupling the linker via click chemistry. Forexample, the nucleosides shown below can be used to synthesize sucholigonucleotides:

Exemplary reagents which allow linking targeting oligonucleotidesthrough a nucleobase include protected amino functionality at the basethat can then be coupled to other suitable functional groups. In certainembodiments, Fmoc Amino-Modifier C6 dT (Glen Research catalog number10-1536-xx) is used as a starting material:

Other exemplary reagents which allow linking targeting oligonucleotidesthrough a nucleobase include protected thiol functionality at the basethat can then be coupled to other suitable functional groups or used toform a disulfide bond. In certain embodiments, S-Bz-Thiol-Modifier C6 dT(Glen Research catalog number 10-1039-xx) is used as a startingmaterial:

In other embodiments, Amino-Modifier Serinol Phosphoramidite (GlenResearch catalog number 10-1997-xx) or 3′-Amino-Modifier Serinol CPG(Glen Research catalog number 20-2997-xx) is used to introduceamino-functionalized linkers that can then be coupled with othersuitable functional groups:

In other embodiments, Thiol-Modifier C6 S—S(Glen Research catalog number10-1936-xx), 3′-Thiol-Modifier C3 S—S CPG (Glen Research catalog number20-2933-xx), or 5′-Maleimide-Modifier Phosphoramidite (Glen Researchcatalog number 10-1938-xx) is used to introduce a linker:

In some embodiments, Cholesteryl-TEG Phosphoramidite (Glen Researchcatalog number 10-1975-xx) or α-Tocopherol-TEG Phosphoramidite (GlenResearch catalog number 10-1977-xx) is used in phosphoramidite synthesisto add a lipophilic moiety to an targeting oligonucleotide:

In some embodiments, one or more of the following starting materials areused in oligonucleotide synthesis to introduce an alkyne into antargeting oligonucleotide that can be reacted via click chemistry withan azide to attach another targeting oligonucleotide or another moietysuch as a lipophilic group or targeting group:

In some embodiments, one or more of the following starting materials areused to attach a lipophilic group or targeting group via click chemistryto an targeting oligonucleotide functionalized with an alkyne, such asthe ones described above:

E. Targets and Uses

The disclosure provides a method of inhibiting target expression levelsof one or more targets, comprising administering to a cell or a subjectthe compounds of the invention in an amount effective to inhibit theexpression of the target(s). In certain embodiments, the target is anmRNA. In other embodiments, the target could be a microRNA, as describedabove. In such cases, the individual targeting oligonucleotides may bereferred to as “antagomiRs.” In other embodiments, the target can be anon-coding RNA naturally expressed in the cells.

The subjects treated according to the methods of the invention can beanimals, including humans, primates, and rodents. Cells can be presentin vitro, or treated ex vivo. In some cases, ex vivo treated cells arere-administered to the subject.

The invention also encompasses dual and multiple target antisenseinhibitors, in particular those to treat liver diseases, metabolicdiseases, cardiovascular diseases, inflammatory diseases, neurologicaldiseases, viral, bacterial, parasitic, or prion infections and cancer.In particular, it includes the use of dimeric antisense inhibitors toinhibit liver targets (also referred to as “hepatic targets”), such asApoB and ApoC3 dual inhibition. Since knock-down of ApoB has beenreported to lead to undesired lipid deposition in the liver, thesimultaneous knock-down of ApoC3 can decrease this side effect.

In cancer, the simultaneous knock-down of two targets can lead tosynergistic anti-tumor effects. In particular, combination of targetswith different mechanisms of action and signaling pathways should be ofinterest, e.g., a combination of cytostatic mechanism withanti-metastatic mechanism.

By selecting appropriate sequences against various cancer or tumorrelated targets, the present invention is also suitable for cancertreatment. Thus, it is possible to use multimeric oligonucleotidecompounds of the invention that comprise targeting oligonucleotideswhich are directed 1) against targets responsible for thedifferentiation, development, or growth of cancers, such as:oncoproteins or transcription factors, e.g., c-myc, N-myc, c-myb, c-fos,c-fos/jun, PCNA, p120, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, bcl-x,bcl-w, cdc-2, c-raf-1, c-mos, c-src, c-abl, c-ets; 2) against cellularreceptors, such as EGF receptor, Her-2, c-erbA, VEGF receptor (KDR-1),retinoid receptors; 3) against protein kinases, c-fms, Tie-2, c-raf-1kinase, PKC-alpha, protein kinase A (R1 alpha); 4) against growth orangiogenic factors, such as bFGF, VEGF, EGF, HB-EGF, PDGF and TGF-β3; 5)against cytokines, such as IL-10, against cell cycle proteins, such ascyclin-E; 6) against tumor proteins, such as MAT-8; or 7) againstinhibitors of tumor suppressor genes such as MDM-2. Also of use areantisense or directed against 8) components of spindle formation, suchas eg5 and PLK1, or 9) against targets to suppress metastasis, such asCXCR4. Of use are antisense sequences directed against 10) factors whichsuppress apoptosis, such as survivin, stat3 and hdm2, or which suppressthe expression of multiple drug resistance genes, such as MDR1(P-glycoprotein).

The dimer/multimer can also degrade or antagonize microRNA (miRNA) whichare single-stranded RNA molecules of about 21-23 nucleotides in lengthregulating gene expression. miRNAs are encoded by genes that aretranscribed from DNA but not translated into protein (non-coding RNA);instead they are processed from primary transcripts known as pri-miRNAto short stem-loop structures called pre-miRNA and finally to functionalmiRNA. Mature miRNA molecules are partially complementary to one or moremessenger RNA (mRNA) molecules, and their main function is todown-regulate gene expression. It appears that many miRNA sequencesdiscovered in the human genome contribute to the development of cancer.Some miRNAs are significantly deregulated in cancer. Further, miRNAwhich is over-expressed (e.g., TGF-β2 receptor, RB 1 and PLAG1) leadingto tumor growth can be down-regulated using antisense approaches asdescribed before. An miRNA expression signature of human solid tumorsdefining cancer gene targets was reported, for example, by Volinia etal., PNAS, 2006, 103, 2257-61.

Further provided are pharmaceutical compositions, comprising a compoundof the invention and one or more pharmaceutically acceptable excipients.Methods of formulating and administering oligonucleotides to a cell or asubject are known in the art (see, e.g., Hardee, Gregory E.; Tillman,Lloyd G.; Geary, Richard S. Routes and Formulations For Delivery ofAntisense Oligonucleotides. Antisense Drug Technology (2nd Edition)2008, 217-236. Publisher: CRC Press LLC, Boca Raton, Fla.; Zhao et al.,2009, Expert Opin. Drug Deliv., 6:673-686; Juliano et al., 2008, NucleicAcids Res., 36:4158-4171; Augner, 2006, J. Biomed. Biotechnol. 1-15;Wilson et al., 2005, Advances Genetics, 54:21-41; Hassane et al., 2010,Cell. Mol. Life Sci., 67:715-726; and Nakagawa et al., 2010, J. Am.Chem. Soc., 132:8848-8849.

In some embodiments, the compounds of the invention possess favorablepharmacokinetic and/or pharmacodynamic properties. For example, in somecase, a therapeutically effective knockdown of the target(s) persistsfor two weeks or longer following the administration. In someembodiments, the compositions of the invention are characterized by oneor more of the following properties when administered in vivo:

(d) increased concentration in the liver (or other tissues) and reducedclearance by kidneys as compared to respective monomeric targetingoligonucleotides;

(e) longer duration of target knockdown as compared to respectivemonomeric targeting oligonucleotides; and

(f) lower effective concentrations as compared to respective monomerictargeting oligonucleotides and/or the same multimeric oligonucleotidecompound, wherein the cleavable linker is substituted with anoncleavable linker.

F. Routes of Delivery

A composition that includes a multimeric oligonucleotide compound can bedelivered to a subject by a variety of routes. Exemplary routes include:intravenous, intradermal, topical, rectal, parenteral, anal,intravaginal, intranasal, pulmonary, ocular. The term “therapeuticallyeffective amount” is the amount of multimeric oligonucleotide compoundpresent in the composition that is needed to provide the desired levelof target gene modulation (e.g., inhibition or activation) in thesubject to be treated to give the anticipated physiological response.The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect. The term“pharmaceutically acceptable carrier” means that the carrier can beadministered to a subject with no significant adverse toxicologicaleffects to the subject.

The multimeric oligonucleotide compound molecules of the invention canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically include one or more speciesof multimeric oligonucleotide compounds and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the multimeric oligonucleotide compound inaerosol form. The vascular endothelial cells could be targeted bycoating a balloon catheter with the multimeric oligonucleotide compoundand mechanically introducing the oligonucleotide.

Topical administration refers to the delivery to a subject by contactingthe formulation directly to a surface of the subject. The most commonform of topical delivery is to the skin, but a composition disclosedherein can also be directly applied to other surfaces of the body, e.g.,to the eye, a mucous membrane, to surfaces of a body cavity or to aninternal surface. As mentioned above, the most common topical deliveryis to the skin. The term encompasses several routes of administrationincluding, but not limited to, topical and transdermal. These modes ofadministration typically include penetration of the skin's permeabilitybarrier and efficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.Topical administration can also be used as a means to selectivelydeliver oligonucleotides to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy. In addition, iontophoresis (transferof ionic solutes through biological membranes under the influence of anelectric field), phonophoresis or sonophoresis (use of ultrasound toenhance the absorption of various therapeutic agents across biologicalmembranes, notably the skin and the cornea), and optimization of vehiclecharacteristics relative to dose position and retention at the site ofadministration may be useful methods for enhancing the transport oftopically applied compositions across skin and mucosal sites.

Both the oral and nasal membranes offer advantages over other routes ofadministration. For example, oligonucleotides administered through thesemembranes may have a rapid onset of action, provide therapeutic plasmalevels, avoid first pass effect of hepatic metabolism, and avoidexposure of the oligonucleotides to the hostile gastrointestinal (GI)environment. Additional advantages include easy access to the membranesites so that the oligonucleotide can be applied, localized and removedeasily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many agents. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

A pharmaceutical composition of multimeric oligonucleotide compound mayalso be administered to the buccal cavity of a human being by sprayinginto the cavity, without inhalation, from a metered dose spraydispenser, a mixed micellar pharmaceutical formulation as describedabove and a propellant. In one embodiment, the dispenser is first shakenprior to spraying the pharmaceutical formulation and propellant into thebuccal cavity.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, slurries, emulsions, elixirsor non-aqueous media, tablets, capsules, lozenges, or troches. In thecase of tablets, carriers that can be used include lactose, sodiumcitrate and salts of phosphoric acid. Various disintegrants such asstarch, and lubricating agents such as magnesium stearate, sodium laurylsulfate and talc, are commonly used in tablets. For oral administrationin capsule form, useful diluents are lactose and high molecular weightpolyethylene glycols. When aqueous suspensions are required for oraluse, the nucleic acid compositions can be combined with emulsifying andsuspending agents. If desired, certain sweetening and/or flavoringagents can be added.

Parenteral administration includes intravenous drip, subcutaneous,intraperitoneal or intramuscular injection, intrathecal orintraventricular administration. In some embodiments, parentaladministration involves administration directly to the site of disease(e.g. injection into a tumor).

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

Any of the multimeric oligonucleotide compounds described herein can beadministered to ocular tissue. For example, the compositions can beapplied to the surface of the eye or nearby tissue, e.g., the inside ofthe eyelid. For ocular administration, ointments or droppable liquidsmay be delivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers. The multimeric oligonucleotide compound canalso be administered to the interior of the eye, and can be introducedby a needle or other delivery device which can introduce it to aselected area or structure.

Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably multimericoligonucleotide compounds, within the dispersion can reach the lungwhere it can be readily absorbed through the alveolar region directlyinto blood circulation. Pulmonary delivery can be effective both forsystemic delivery and for localized delivery to treat diseases of thelungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver agents that may be readily formulated as dry powders. Amultimeric oligonucleotide compound may be stably stored as lyophilizedor spray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 a m. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred. Pulmonary administration of amicellar multimeric oligonucleotide compound formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Exemplary devices include devices which are introduced into thevasculature, e.g., devices inserted into the lumen of a vascular tissue,or which devices themselves form a part of the vasculature, includingstents, catheters, heart valves, and other vascular devices. Thesedevices, e.g., catheters or stents, can be placed in the vasculature ofthe lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to amultimeric oligonucleotide compound, e.g., a device can release insulin.

In one embodiment, unit doses or measured doses of a composition thatincludes multimeric oligonucleotide compound are dispensed by animplanted device. The device can include a sensor that monitors aparameter within a subject. For example, the device can include pump,e.g., and, optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with a multimericoligonucleotide compound, ex vivo and then administered or implanted ina subject. The tissue can be autologous, allogeneic, or xenogeneictissue. E.g., tissue can be treated to reduce graft v. host disease. Inother embodiments, the tissue is allogeneic and the tissue is treated totreat a disorder characterized by unwanted gene expression in thattissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrowhematopoietic cells, can be treated to inhibit unwanted cellproliferation. Introduction of treated tissue, whether autologous ortransplant, can be combined with other therapies. In someimplementations, the multimeric oligonucleotide compound treated cellsare insulated from other cells, e.g., by a semi-permeable porous barrierthat prevents the cells from leaving the implant, but enables moleculesfrom the body to reach the cells and molecules produced by the cells toenter the body. In one embodiment, the porous barrier is formed fromalginate.

In one embodiment, a contraceptive device is coated with or contains amultimeric oligonucleotide compound. Exemplary devices include condoms,diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths,and birth control devices.

G. Dosage

In one aspect, the invention features a method of administering amultimeric oligonucleotide compound to a subject (e.g., a humansubject). In one embodiment, the unit dose is between about 10 mg and 25mg per kg of bodyweight. In one embodiment, the unit dose is betweenabout 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unitdose is between about 0.1 mg and 500 mg per kg of bodyweight. In someembodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05, 0.1,0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with aparticular target gene. The unit dose, for example, can be administeredby injection (e.g., intravenous or intramuscular), an inhaled dose, or atopical application.

In some embodiments, the unit dose is administered daily. In someembodiments, less frequently than once a day, e.g., less than every 2,4, 8 or 30 days. In another embodiment, the unit dose is notadministered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. In someembodiments, the unit dose is administered more than once a day, e.g.,once an hour, two hours, four hours, eight hours, twelve hours, etc.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a multimeric oligonucleotide compound. Themaintenance dose or doses are generally lower than the initial dose,e.g., one-half less of the initial dose. A maintenance regimen caninclude treating the subject with a dose or doses ranging from 0.0001 to100 mg/kg of body weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or0.0001 mg per kg of bodyweight per day. The maintenance doses may beadministered no more than once every 1, 5, 10, or 30 days. Further, thetreatment regimen may last for a period of time which will varydepending upon the nature of the particular disease, its severity andthe overall condition of the patient. In some embodiments the dosage maybe delivered no more than once per day, e.g., no more than once per 24,36, 48, or more hours, e.g., no more than once for every 5 or 8 days.Following treatment, the patient can be monitored for changes in hiscondition and for alleviation of the symptoms of the disease state. Thedosage of the oligonucleotide may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.0001 mg to 100 mg per kg of bodyweight.

The concentration of the multimeric oligonucleotide compound is anamount sufficient to be effective in treating or preventing a disorderor to regulate a physiological condition in humans. The concentration oramount of multimeric oligonucleotide compound administered will dependon the parameters determined for the agent and the method ofadministration, e.g. nasal, buccal, pulmonary. For example, nasalformulations may tend to require much lower concentrations of someingredients in order to avoid irritation or burning of the nasalpassages. It is sometimes desirable to dilute an oral formulation up to10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a multimeric oligonucleotidecompound can include a single treatment or, preferably, can include aseries of treatments. It will also be appreciated that the effectivedosage of a multimeric oligonucleotide compound used for treatment mayincrease or decrease over the course of a particular treatment. Forexample, the subject can be monitored after administering a multimericoligonucleotide compound. Based on information from the monitoring, anadditional amount of the multimeric oligonucleotide compound can beadministered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of target gene expression levels in thebody of the patient. Persons of ordinary skill can easily determineoptimum dosages, dosing methodologies and repetition rates. Optimumdosages may vary depending on the relative potency of individualcompounds, and can generally be estimated based on EC50s found to beeffective in in vitro and in vivo animal models. In some embodiments,the animal models include transgenic animals that express a human targetgene. In another embodiment, the composition for testing includes amultimeric oligonucleotide compound that is complementary, at least inan internal region, to a sequence that is conserved between target genein the animal model and the target gene in a human.

In one embodiment, the administration of the multimeric oligonucleotidecompound is parenteral, e.g. intravenous (e.g., as a bolus or as adiffusible infusion), intradermal, intraperitoneal, intramuscular,intrathecal, intraventricular, intracranial, subcutaneous, transmucosal,buccal, sublingual, endoscopic, rectal, oral, vaginal, topical,pulmonary, intranasal, urethral or ocular. Administration can beprovided by the subject or by another person, e.g., a health careprovider. The composition can be provided in measured doses or in adispenser which delivers a metered dose. Selected modes of delivery arediscussed in more detail below.

H. Kits

In certain aspects of the invention, kits are provided, comprising acontainer housing a composition comprising a multimeric oligonucleotidecompound. In some embodiments, the composition is a pharmaceuticalcomposition comprising a multimeric oligonucleotide compound and apharmaceutically acceptable carrier. In some embodiments, the individualcomponents of the pharmaceutical composition may be provided in onecontainer. Alternatively, it may be desirable to provide the componentsof the pharmaceutical composition separately in two or more containers,e.g., one container for multimeric oligonucleotide compounds, and atleast another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

The following examples provide illustrative embodiments of theinvention. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present invention. Such modifications andvariations are encompassed within the scope of the invention. TheExamples do not in any way limit the invention.

EXAMPLES Example 1: Design of Antisense Oligonucleotides

Antisense oligonucleotides against ApoC3, ApoB, Hif-1alpha, survivin andB2M were either selected using a series of bioinformatics filters andcomputational design algorithms or were derived from the literature.They were selected to be 13, 14 or more nucleotides in length and testedusing one or multiple chemical modification design patterns (forexample, 3LNAs-8DNAs-3LNAs). The list of all targeting oligonucleotidesequences is given in Table 1 and specific chemical modificationpatterns are explicitly specified when data is presented. Factors takeninto account during the design include species homology, alignment tomultiple human transcripts, off-target matches, SNPs, exon-exonboundaries, coverage of the transcript, and statistical models ofefficacy and polyA regions. For species homology human, rat, mouse andmacaque sequences were considered. For off-target matches, putativesequences were searched against the human transcriptome and perfectmatches were identified along with compounds that had only 1 or 2mismatches. Preference was given to compounds with no perfect off-targetmatches, but compounds were selected with 1 or 2 mismatches if thecompound met many of the other criteria. Statistical classificationmodels were derived from existing in-house projects for other targetingoligonucleotide projects. These models were applied to the potentialASOs and preference given to those classified as active. Known SNPs,exon-exon boundaries and polyA regions were also avoided in the designwhen possible. Other ASO design features are also well known in the art,such as avoidance of immune stimulatory sequences such as CpG motifs,avoidance of poly G regions, and avoidance of toxic sequences such ascertain poly-pyrimidine motifs.

FIGS. 1A and 1B show schematic representation of exemplarydimeric/multimeric constructs. Specifically, in FIG. 1A, two 14-mergapmers (e.g., 3LNA-8DNA-3LNA) are connected via a cleavable linker,which can be cleaved by enzymes, such as nucleases, peptidases or byreduction or oxidation. It could also be a linker which is cleaved by apH shift within the cells (e.g. acidic pH in endosomes). The twoantisense gapmers can be identical (homo-dimer), which leads tosuppression of a single target mRNA1. However, the two antisense gapmerscan also have different sequences (hetero-dimer) which are complementaryto two or more different targets and which will lead to inhibition oftwo targets (mRNA1 and mRNA2) or trimers or tetramers, etc., specific to3, 4, or more different targets.

Example 2: Synthesis of Antisense Oligonucleotides (A) General Procedurefor Oligomer Synthesis

All oligonucleotides were synthesized using standard phosphoramiditeprotocols (Beaucage, S. L.; Caruthers, M. H. “Deoxynucleosidephosphoramidites—A new class of key intermediates fordeoxypolynucleotide synthesis”. Tetrahedron Lett., 1981, 22:1859) on aMerMade 192 oligonucleotide synthesizer (BioAutomation) or Oligopilot 10synthesizer (GE) at 200 to 1000 nmole scales employing standard CPGsupports (BioSearch) or Glen UnySupport (Glen Research). The DNA,2′-OMe, 2′-F, and G-clamp monomers were obtained from ChemGenesCorporation or Glen Research, and the LNA monomers were obtained fromother commercially available sources. All phosphoramidites other thanDNA were coupled with extended coupling times (e.g. 8 to 15 min for RNA,LNA, 2′-O-Methyl, 2′-Fluoro, 5-Propynyl and G-Clamps). After thesynthesis, the oligonucleotides were cleaved from the support anddeprotected using AMA (a 50:50 mixture of ammonium hydroxide and aqueousmethylamine) at 65° C. for one hour or using aqueous ammonium hydroxideat 55° C. for 8 hours. The crude DMTr-on oligonucleotides were purifiedvia DMTr-selective cartridge purification techniques and if necessaryfurther purified via RP HPLC and desalted via cartridge-based methods.Alternatively, they were purified using ion exchange chromatography. Thefinal oligonucleotides were characterized using LC-MS.

A C Technologies Solo VP Slope (Bridgewater, N.J.) reader equipped with“Quick Slope” software was used to determine the concentration ofoligonucleotides. Fifty μl of sample was required for the measurement ina micro quartz vessel. The instrument measured the change in absorbanceat varying path lengths, utilizing Beer's Law to determine finalconcentrations. Extinction coefficients were calculated using thenearest neighbor model.

(B) Synthesis of Linear Dimers and Trimers

The synthesis of linear dimers and trimers was completed by linearaddition of all monomers until the full length sequence was obtained onsolid support. First, ASO 1 was completely synthesized followed byaddition of the cleavable or noncleavable linker X (e.g., tri-thymidyl,tetra-thymidyl, tetra-uridyl, disulfide, etc.) and finally either ASO 1(“homo”-dimer) or an ASO with another sequence ASO 2 (“hetero”-dimer)and optionally directed against another target mRNA was added. The ASOsynthesized first is connected to the linker X via its 5′-end whereasthe finally synthesized ASO is connected via 3′. This might lead to 3′-and 5′-modified metabolites after cleavage. Due to its linearity, atrimer, tetramer, or other multimer could be synthesized by adding asecond, third, or more cleavable linker(s) followed by another ASO (1, 2or 3).

As illustrated above, for example, SED ID NO:2 (ApoC3-ApoC3 homodimerASO) contains ASO1 (βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG (SEQ IDNO: 1)), with X being dT-dT-dT (tri-thymidyl), wherein “—” is aphosphodiester linkage and “*” is a phosphorothioate linkage, dN is2′-deoxynucleotide and, βN is an LNA nucleotide).

A general example for a linear trimer is given below:

(C) Synthesis of 3′3′-branched Dimers (Doubler Dimers)

For symmetric dimers, synthesis was performed using a triethylene glycol(teg) derivatized solid support and a symmetric doubler (brancher)phosphoramidite from Glen Research (catalog number 10-1920) illustratedbelow

After coupling of the brancher phosphoramidite (catalog No. 10-1920) tothe triethylene glycol bound to the solid phase, the DMT protectinggroups were removed with acid and coupling of linker X and ASO 1 wasperformed in parallel as illustrated in FIG. 1C. For SEQ ID NO:4(βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG-dT-dT-dT-dT-)2doub*teg,linker X is dT-dT-dT-dT, Y is Oxygen, * is phosphorothioate, — isphosphodiester, and “doub*teg” stands for the following substructure:

For the synthesis of two identical strands in parallel a double-couplingstep was performed on the oligonucleotide synthesizer to yield maximumcoupling efficiency on both strands.

With an asymmetric doubler phosphoramidite (Glen Research, catalognumber 10-1921) the synthesis of “hetero”-dimers is possible. Thus, ASO1 is connected first to the doubler and ASO 2 is connected second.

The symmetrical doubler or branching strategy was also performed withglycerol like CPG solid support from Chemgenes (N-5216-05 andN-7170-05). Symmetrical branching doubler N-5216-05 is shown in FormulaVI.

The asymmetrical branching doubler N-7170-05 is shown in Formula VII.

Oligonucleotide dimers synthesized with this doubler (brancher) have thefollowing structure:

(D) Synthesis of Branched Trimers

For the synthesis of trimers with two different ASO molecules, the firstASO1 is synthesized by linear addition of all monomers until the fulllength sequence of ASO1 was obtained on solid support. Then, thecleavable (or noncleavable) linker X (tetrathymidyl, tetrauridyl,disulfide etc.) is synthesized followed by addition of the doubler usinga symmetric doubler phosphoramidite (Glen Research, 10-1920) the solidphase synthesis of the linker X and ASO 1 was performed in parallel asindicated in the figure below.

After removal of the DMT protecting groups of the symmetric doubler, twoASO2 molecules are synthesized simultaneously from 3′ to 5′ directionusing standard phosphoramidite chemistry. This results in a trimerconsisting of two ASO2 molecules having two free 5′-ends and one ASO1molecule having one free 3′-end of the structure shown in FIG. 1D.

For the synthesis of branched trimers consisting of three differentASO1, ASO2 and ASO3 molecules, the non-symmetric brancherphosphoramidite is coupled after first synthesis of ASO1, followed bysequential synthesis of ASO2 and ASO3. The non-symmetricalphosphoramidite structure is shown in Formula IX. The resulting trimerhas the structure show in FIG. 1D.

It is also possible to use a “trebler” phosphoramidite (Glen Research,10-1922) shown in Formula X which results in symmetrical homo trimers.

(E) Sequences of Synthesized Oligonucleotides and Characterization byMass Spectrometry

All compounds were purified by IEX HPLC or IP-RP HPLC and characterizedusing LC-MS methods. The following listing in Table 1 provides specificsequences and modification patterns with the corresponding SEQ ID NOs onthe left followed by a detailed description.

TABLE 1 SEQ Reference ID Sequence NO: Description 1 103966βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG3LNA-8DNA-3LNA gapmer (monomeric), fully phosphorothioated ApoC3 2105360 βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG-dT-dT-dT-βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βGlinear homodimer from SEQ ID NO: 1 with 3 nt phosphodiester linker ApoC33 105361βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG*teg*SS*teg*βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βGlinear homodimer from SEQ ID NO: 1 with disulfide linker ApoC3 4105362 ((βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG-dT-dT-dT-dT-)₂doub*teg3′3′-branched homodimer from SEQ ID NO: 1 with 2 ×4 nt phosphodiester linker ApoC3 5 105363(βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG*teg*SS*)₂doub*teg3′3′-branched homodimer from SEQ ID NO: 1 with disulfide linker ApoC3 6105395 βA*βA*βG*dC*dA*dA*dC*dC*dT*dT*dC*βA*βG*βG-dT-dT-dT-βA*βA*βG*dC*dA*dA*dC*dC*dT*dT*dC*βA*βG*βG mouse ortholog of SEQ ID NO: 2ApoC3 7 105513 βA*βA*βG*dC*dA*dA*dC*dC*dT*dT*dC*βA*βG*βG-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer (from SEQ ID NOs: 13/14) with 3 nt phosphodiester-linkerApoB/ApoC3 (mouse) 8 105514βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-βT*βZ*βZ*dT*dC*dG*dG*dC*dC*dT*βZ*βT*βGlinear heterodimer (from SEQ ID NOs: 13/10) with 3 nt phosphodiester-linkerApoB/ApoC3 9 104109 βZ*βZ*βT*dC*dT*dT*dC*dG*dG*dC*dC*βZ*βT*βG3LNA-8DNA-3LNA gapmer (monomeric), fully phosphorothioated ApoB 10104111 βT*βZ*βZ*dT*dC*dG*dG*dC*dC*dT*βZ*βT*βG3LNA-7DNA-3LNA gapmer (monomeric), fully phosphorothioated ApoC3 11104112 βT*βZ*βT*dT*dC*dG*dG*dC*dC*dC*βT*βG3LNA-7DNA-2LNA gapmer (monomeric), fully phosphorothioated ApoB 12105576 βT*βZ*βT*dT*dZ*dG*dG*dC*dC*dC*βT*βG5-methyl-dC (dZ) analog of SEQ ID NO: 11 ApoB 13 102102βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA2LNA-8DNA-3LNA gapmer (monomeric), fully phosphorothioated ApoB 14105515 βA*βA*βG*dC*dA*dA*dC*dC*dT*dT*dC*βA*βG*βGmouse ortholog of SEQ ID NO: 1 ApoC3 15 106200βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βTlinear homodimer from SEQ ID NO: 30 with 4 nt phosphodiester DNA linkerApoC3 16 106201βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT*dT*dT*dT*dT*βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βTlinear homodimer from SEQ ID NO: 30 with 4 nt phosphorothioate DNA linkerApoC3 17 106202 (βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-)₂doub*teg 3′3′-branched homodimer from SEQ ID NO: 30, 2 ×4 nt phosphodiester DNA linker ApoC3 18 106203βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer from SEQ ID NO: 13 with phosphodiester DNA linker ApoB19 106204βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA*dT*dT*dT*dT*βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer from SEQ ID NO: 13 with phosphorothioate DNA linkerApoB 20 106205(βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-)₂doub*teg3′3′-branched homodimer from SEQ ID NO: 13, phosphodiester DNA linkerApoB 21 106206 βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 30/13 with phosphodiester DNA linkerApoC3/ApoB 22 106207 βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βTlinear heterodimer from SEQ ID NO: 13/30 with phosphodiester DNA linkerApoB/ApoC3 23 106413 βG*βG*dC*dA*dA*dG*dC*dA*dT*dC*βZ*βT*βG-dT-dT-dT-dT-βZ*βA*dA*dT*dC*dC*dA*dT*dG*dG*βZ*βA*βGlinear heterodimer from SEQ ID NO: 27/28 with phosphodiester DNA linkerHIF-1alpha/survivin 24 106414βG*βG*dC*dA*dA*dG*dC*dA*dT*dC*βZ*βT*βG-dT-dT-dT-dT-βG*βZ*βG*dT*dG*dC*dA*dT*dA*dA*dA*βT*βT*βGlinear heterodimer from SEQ ID NO: 27/29 with phosphodiester DNA linkerHIF-1alpha/B 2M 25 106415βG*βG*dC*dA*dA*dG*dC*dA*dT*dC*βZ*βT*βG-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 27/13 with phosphodiester DNA linkerHIF-1alpha/Apo B 26 106416βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-βG*βG*dC*dA*dA*dG*dC*dA*dT*dC*βZ*βT*βGlinear heterotrimer from SEQ ID NO: 13/30/27 with two phosphodiester DNA linkersApoB/ApoC3/HIF-1alpha 27 101443 βG*βG*dC*dA*dA*dG*dC*dA*dT*dC*βZ*βT*βG2LNA-8DNA-3LNAgapmer (monomeric), fully phosphorothioated HIF-1alpha 28101441 βZ*βA*dA*dT*dC*dC*dA*dT*dG*dG*βZ*βA*βG2LNA-8DNA-3LNAgapmer (monomeric), fully phosphorothioated Survivin 29105758 βG*βZ*βG*dT*dG*dC*dA*dT*dA*dA*dA*βT*βT*βG3LNA-8DNA-3LNAgapmer (monomeric), fully phosphorothioated B2M 30 104975βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT2LNA-10DNA-2LNAgapmer (monomeric), fully phosphorothioated ApoC3 31102103 βZ*βG*dT*dC*dT*dA*dT*dG*dT*dA*βT*βA*βG2LNA-8DNA-3LNA gapmer (monomeric), fully phosphorothioatedApoB negative control (mismatched) 32 104882mU*mU*APC*dA*dG*dT*dG*dT*dG*dA*dT*mG*mA*APC2me-9DNA-2me gapmer with 2 G-clamps (APC), fully phosphorothioated (monomeric),ApoC3 33 106417 βZ*βZ*mA*dG*dT*dA*dG*dT*dC*dT*dT*mU*βZ*βA-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 55/13 with phosphodiester DNA linkerApoC3/ApoB 34 106418βZ*βZ*mA*dG*dT*dA*dG*dT*dC*dT*dT*mU*mC*mA-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 56/13 with phosphodiester DNA linkerApoC3/ApoB 35 106419βZ*βZ*fA*dG*dT*dA*dG*dT*dC*dT*dT*fU*fC*fA-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 57/13 with phosphodiester DNA linkerApoC3/ApoB 36 106420βG*βG*βA*βA*dC*dT*dG*dA*dA*dG*dC*dC*dA*dT-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 58/13 with phosphodiester DNA linker, (5′nucleotide can also be substitute with a G) ApoC3/ApoB 37 106206βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 30/13 with phosphodiester DNA linker, (5′nucleotide can also be substitute with a G) ApoC3/ApoB 38 106421mU*mU*APC*dA*dG*dT*dG*dT*dG*dA*dT*mG*mA*APC-dT-dT-dT-dT-βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βTlinear heterodimer from SEQ ID NO: 32/13 with phosphodiester DNA linkerApoC3/ApoB 39 106422βA*βA*βG*dC*dA*dA*dC*dC*dT*dA*dC*βA*βG*βG-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear heterodimer from SEQ ID NO: 1/13 with phosphodiester DNA linkerApoC3/ApoB 40 106423(βG*βG*βA*βA*dC*dT*dG*dA*dA*dG*dC*dC*dA*dT-dT-dT-dT-dT)₂doub*teg 3′3′-branched homodimer from SEQ ID NO: 58, phosphodiester DNA linker ApoC341 106424 βG*βZ*dA*PC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-βG*βZ*dA*PC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βTlinear homodimer with 5-propynyl-dC with phosphodiester DNA linker ApoC342 106425 βG*βZ*dA*PC*PU*dG*dA*dG*dA*dA*dT*dA*βZ*βT-dT-dT-dT-dT-βG*βZ*dA*PC*PU*dG*dA*dG*dA*dA*dT*dA*βZ*βTlinear homodimer with 5-propynyl-dC/dU and with phosphodiester DNA linkerApoC3 43 106426 βG*βZ*dA*PC*PU*dG*dA*dG*dA*dA*PU*dA*βZ*βT-dT-dT-dT-dT-βG*βZ*dA*PC*PU*dG*dA*dG*dA*dA*PU*dA*βZ*βTlinear homodimer with 5-propynyl-dC/dU and with phosphodiester DNA linkerApoC3 44 106234 βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 3 phosphodiester linkages in the 2 nt DNAlinker ApoB 45 106235 βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 4 phosphodiester linkages in the 3 nt DNAlinker ApoB 46 106236βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 5 phosphodiester linkages in the 4 nt DNAlinker ApoB 47 106237βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 6 phosphodiester linkages in the 5 nt DNAlinker ApoB 48 106238βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 7 phosphodiester linkages in the 6 nt DNAlinker ApoB 49 106239βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-dT-dT-dT-dT-dT-dT-dT-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 8 phosphodiester linkages in the 7 nt DNAlinker ApoB 50 106241βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA*dT*dT-dT-dT-dT-dT*dT*βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 4 phosphodiester/4 phosphorothioate linkagesin the 7 nt DNA linker ApoB 51 106242βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA*rU*βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 2 phosphorothioate linkages in the 1 nt RNAlinker ApoB 52 106243βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA*rU*rU*βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 3 phosphorothioate linkages in the 2 nt RNAlinker ApoB 53 106244βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA*rU*rU*rU*βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 4 phosphorothioate linkages in the 3 nt RNAlinker ApoB 54 106245βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA*rU*rU*rU*rU*βG*βZ*dA*Dt*dT*dG*dG*dT*dA*dT*βT*βZ*βAlinear homodimer of SEQ ID NO: 13 with 5 phosphorothioate linkages in the 4 nt RNAlinker ApoB 55 105448 βZ*βZ*mA*dG*dT*dA*dG*dT*dC*dT*dT*mU*βZ*βA2LNA-1me-8DNA-1me-2LNA  ApoC3 56 105382βZ*βZ*mA*dG*dT*dA*dG*dT*dC*dT*dT*mU*mC*mA2LNA-1me-8DNA-3me gapmer (monomeric), fully phosphorothioated ApoC3 57105390 βZ*βZ*fA*dG*dT*dA*dG*dT*dC*dT*dT*fU*fC*fA2LNA-1fluoro-8DNA-3fluoro gapmer (monomeric), fully phosphorothioatedApoC3 58 105704 βG*βG*βA*βA*dC*dT*dG*dA*dA*dG*dC*dC*dA*dT4LNA-10DNA antisense (monomeric), fully phosphorothioated ApoC3

Table 2 provides a descriptive legend for chemical structuredesignations used throughout the specification, including in Table 1.

TABLE 2 Designation Description Chemical Structure - (in some contexts)phosphodiester

* phosphorothioate

dA 2′-deoxyadenosine

rA (ribo)adenosine

mA 2′-O-methyl- adenosine

βA LNA [2′,4′]- locked adenosine

fA 2′-fluoro-ribo- adenosine

dC 2′-deoxycytidine

rC (ribo)cytidine

βC LNA [2′,4′]-locked- cytidine

fC 2′-fluoro-ribo- cytidine

dG 2′- deoxyguanosine

rG guanosine

mG 2′-O-methyl- guanosine

βG LNA [2′,3′]-locked guanosine

fG 2′-fluoro-ribo- guanosine

dT 2′-deoxythmidine

mT 2′-O-methyl- “ribo”-thymidine (3′-5′)

βT LNA [2′,4′]-locked “ribo”-thymidine

dU 2′-deoxyuridine

rU uridine

mU 2′-O-methyl- uridine

fU 2′-fluoro-ribo- uridine

βU LNA [2′,4′]-locked uridine

Z 5-methyl-2′- deoxy-cytidine

βZ LNA [2′,4′]-locked 5- methyl-cytidine

APC G-Clamp deoxy- phenoxazine

PC 5-propynyl-2′- deoxycytidine

PU 5-propynyl-2′- deoxythymidine

teg triethylenglycol

doub doubler

(NH2—C12- amino) (NH2—C12- amino)

Spacer 18 Spacer 18

dT-Biotin dT-Bitotin

Bioten TEG Biotin TEG

HEG HEG

The measured molecular weights of the oligonucleotides were tested andfound to be in agreement with the calculated values (see Table 3).

TABLE 3 Ref. Calculated MW by SEQ ID NO: Number MW LC-MS 1 103966 4642.74642.4 2 105360 10260.1 10259.9 3 105361 10164.0 10164.2 4 10536212362.7 12361.7 5 105363 11105.6 11105.3 6 105395 10242.1 10241.9 7105513 9933.9 9933.7 8 105514 9580.6 9580.3 9 104109 4585.7 4585.5 10104111 4280.5 4280.1 11 104112 3913.2 3918 12 105576 4655.9 4655.6 13102102 4325.5 4325.4 14 105515 4633.8 4633.5 15 10620 10520.3 10520.4 16106201 10600.6 10600.4 17 106202 12317.7 12318.5 18 106203 9929.7 9929.819 106204 10010.1 10010.1 20 106205 11727.2 11727.0 21 106206 10225.010225.2 22 106207 10225.0 10225.1 23 106413 9889.7 9889.7 24 10641410279.0 10279.2 25 106415 9910.7 9910.8 26 106416 15810.2 15810.6 27101443 4306.5 4307.3 28 101441 4306.5 4304.5 29 105758 4693.8 4692.7 30104975 4620.8 4620.0 31 102103 4311.5 4310.4 32 104882 4893.1 4893 33106417 10227.0 10227.4 34 106418 10217.0 10217.2 35 106419 10168.910168.8 36 106420 10222.0 10222.3 37 106206 10225.0 10225.2 38 10642110792.6 n.d. 39 106422 10247.0 10247.1 40 106423 12311.6 12311.5 41106424 10596.4 10596.5 42 106425 10644.4 10644.6 43 106426 10692.57 n.d.44 106234 9017.1 9017.1 45 106235 9321.3 9321.1 46 106236 9625.5 9626.147 106237 10233.9 10234.2 48 106238 10538.1 10538.6 49 106239 10842.310842.5 50 106241 10906.6 10906.8 51 106242 9051.2 9051.1 52 1062439373.5 9373.2 53 106244 9695.7 9695.5 54 106245 10017.9 10017.5 55105448 4622.8 4622.6 56 105382 4612.8 4612.6 57 105390 4564.7 4564.4 58105704 4617.8 4617.5

Sequence correlation for the unmodified versions of the sequences(except the linker/bridge) and the respective fully modified sequencesis shown in Table 4.

TABLE 4 SEQ SEQ ID ID NO: * Sequence NO: ** 118 AAGCAACCTACAGG 1 61AAGCAACCTACAGG-T-T-T-AAGCAACCTACAGG 2 62AAGCAACCTACAGGtegSStegAAGCAACCTACAGG 3 63(AAGCAACCTACAGG-T-T-T-T-)₂doubteg 4 64 (AAGCAACCTACAGG-tegSS)₂doubteg 565 AAGCAACCTTCAGG-T-T-T-AAGCAACCTTCAGG 6 66AAGCAACCTTCAGG-T-T-T-GZATTGGTATTZA 7 67GZATTGGTATTZA-T-T-T-TZZTCGGCCTZTG 8 68 ZZTCTTCGGCCZTG 9 69 TZZTCGGCCTZTG10 70 TZTTCGGCCCTG 11 71 TZTTZGGCCCTG 12 72 GZATTGGTATTZA 13 73AAGCAACCTTCAGG 14 74 GZACTGAGAATAZT-T-T-T-T-GZACTGAGAATAZT 15 75GZACTGAGAATAZTTTTTGZACTGAGAATAZT 16 76 (GZACTGAGAATAZT-T-T-T-T-)₂doubteg17 77 GZATTGGTATTZA-T-T-T-T-GZATTGGTATTZA 18 78GZATTGGTATTZATTTTGZATTGGTATTZA 19 79 (GZATTGGTATTZA-T-T-T-T-2doubteg 2080 GZACTGAGAATAZT-T-T-T-T-GZATTGGTATTZA 21 81GZATTGGTATTZA-T-T-T-T-GZACTGAGAATAZT 22 82GGCAAGCATCZTG-T-T-T-T-ZAATCCATGGZAG 23 83GGCAAGCATCZTG-T-T-T-T-GZGTGCATAAATTG 24 84GGCAAGCATCZTG-T-T-T-T-GZATTGGTATTZA 25 85GZATTGGTATTZA-T-T-T-T-GZACTGAGAATAZT- 26 T-T-T-T-GGCAAGCATCZTG 86GGCAAGCATCZTG 27 87 ZAATCCATGGZAG 28 88 GZGTGCATAAATTG 29 89GZACTGAGAATAZT 30 90 ZGTCTATGTATAG 31 91 UU(APC)AGTGTGATGA(APC) 32 92ZZAGTAGTCTTUZA-T-T-T-T-GZATTGGTATTZA 33 93ZZAGTAGTCTTUCA-T-T-T-T-GZATTGGTATTZA 34 94ZZAGTAGTCTTUCA-T-T-T-T-GZATTGGTATTZA 35 95GGAACTGAAGCCAT-T-T-T-T-GZATTGGTATTZA 36 96GZACTGAGAATAZT-T-T-T-T-GZATTGGTATTZA 37 97UU(APC)AGTGTGATGA(APC)-T-T-T-T- 38 GZACTGAGAATAZT 98AAGCAACCTACAGG-T-T-T-T-GZATTGGTATTZA 39 99(GGAACTGAAGCCAT-T-T-T-T-)₂doubteg 40 100 GZA(PC)TGAGAATAZT-T-T-T-T- 41GZA(PC)TGAGAATAZT 101 GZA(PC)(PU)GAGAATAZT-T-T-T-T- 42GZA(PC)(PU)GAGAATAZT 102 GZA(PC)(PU)GAGAA(PU)AZT-T-T-T-T- 43GZA(PC)(PU)GAGAA(PU)AZT 103 GZATTGGTATTZA-T-T-GZATTGGTATTZA 44 104GZATTGGTATTZA-T-T-T-GZATTGGTATTZA 45 105GZATTGGTATTZA-T-T-T-T-GZATTGGTATTZA 46 106GZATTGGTATTZA-T-T-T-T-T-GZATTGGTATTZA 47 107 GZATTGGTATTZA-T-T-T-T-T-T-48 GZATTGGTATTZA 108 GZATTGGTATTZA-T-T-T-T-T-T-T- 49 GZATTGGTATTZA 109GZATTGGTATTZATT-T-T-T-TTGZATTGGTATTZA 50 110 GZATTGGTATTZAUGZATTGGTATTZA51 111 GZATTGGTATTZAUUGZATTGGTATTZA 52 112 GZATTGGTATTZAUUUGZATTGGTATTZA53 113 GZATTGGTATTZAUUUUGZATTGGTATTZA 54 114 ZZAGTAGTCTTUZA 55 115ZZAGTAGTCTTUCA 56 116 ZZAGTAGTCTTUCA 57 117 GGAACTGAAGCCAT 58

In Table 4,

A is adenosine

C is cytidine

G is guanosine

T is thymidine

U is uridine

Z is 5-methyl-cytosine

APC is a G-clamp

PU is 5-propynyl-uridine

PC is 5-propynyl-cytidine and

doubteg is

* Sequence without chemical modifications (except bridge/linker)** The sequence of the fully chemically modified sequence correspondingto SEQ ID NO:*

Example 3: Dimer Stability in Plasma and Cleavage in Liver Homogenates

Stability measurements were performed using 4 different oligonucleotides(including dimers and the monomer, SEQ ID NOs:1, 2, 3, 4).

Briefly, oligos were incubated in 95% plasma of mouse or monkey and in5% liver homogenate at a concentration of 30 μM and at 37° C. Samplesfor measurement were taken after 0, 7, 24 and 48 h of incubation.Samples were subjected to a phenol/chloroform extraction and analyzedusing LC-MS.

In detail, stock solutions with a final concentration of 600 μM and afinal volume of 100 μl have been prepared of all oligonucleotides.Twelve pieces of approximately 50 mg of liver from CD1 mouse (female,Charles River) were added to individual Lysing matrix tubes. Acalculated volume of 1×PBS to give a final concentration of 5% liver(W/W) was added to each of the twelve tubes. All samples werehomogenized using a BioRad Fast prep System. The resulting homogenatesolutions were combined to give about 12 ml of 5% liver homogenate in1×PBS which was subsequently used for incubation.

Plasmas used were a Na-Citrate plasma from female NMRI mice (CharlesRiver) and K-EDTA plasma from male Cynomolgous monkeys (SeralabInternational).

Four samples of each oligo were prepared representing each individualincubation time point (0, 7, 24 and 48 h) in mouse and monkey plasma andin mouse liver homogenate, respectively. In addition, a blank sample anda recovery sample were prepared of each oligo and incubation matrix.Generally, plasma samples were prepared by adding 5 μl of the 600 μMoligo stock solution to 95 μl of mouse or monkey plasma, respectively,with a final oligo concentration of 30 μM. Recovery samples wereprepared by adding 5 μl of water to 95 μl of plasma. Blank samples areoligo in water with a final concentration of 100 μM. Liver samples andrecoveries were prepared in the same way except that liver homogenate inPBS was used instead of plasma.

All samples and recoveries were incubated at 37° C. Samples were cooledto room temperature after 0, 7, 24 and 48 h and was subjected tophenol/chloroform purification. To that end, 370 μl of ammoniumhydroxide (15%), 10 μl dithiothreitol (DTT, 1 M, Sigma Cat. No. 43816)and 800 μl premixed phenol/chloroform/isoamyl alcohol (Sigma P2069) wasadded to each sample. Samples were then vortexed for 10 min at a maximumvortex speed and incubated at 4° C. for 20 min. The samples were thencentrifuged at 3500 RFC for 20 min at 4° C. and 400 μl of the aqueouslayer were removed and dried in a lyophilizer.

The dried samples were dissolved in water (100 μl). The recovery sampleswere dissolved in water (95 μl) and spiked with 5 μl of the respectiveoligo stock solution (600 μM).

Samples were analyzed by LC-MS (Agilent 1200, Bruker Esquire 3000) usinga Waters Acquity UPLC OST C18 column (1.7 μm, 2.1×50) withHFIP/TEA/water (385 mM 1,1,1,3,3,3-hexafluoroisopropanol, 14.4 mMtriethylamine in water) as buffer A and methanol as buffer B at a flowrate of 0.3 ml/min and a column temperature of 60° C. The followinggradient was used: 3 min at 5% B, 5-15% B in 2.5 min (10%/min), 15-23% Bin 5.5 min, 23-30% B in 3 min, 30-100% B in 0.5 min, 5 min at 100% B,100-5% B in 0.5 min, 5 min at 5% B.

Samples were analyzed in 96-well plate format. A standard curve with 8standards (5, 10, 15, 20, 50, 75, 90, 100 μg/ml; 25 μg/ml IS), standard0 (0 μg/ml; 25 μg/ml IS) and three recovery samples (20, 50, 100 μg/ml;25 μg/ml IS) were prepared for each oligo. Samples related to one oligowere analyzed together on the same plate.

Standards were prepared as follows. A piece of approximately 50 mg oftissue was cut from the respective organ tissue, weighted and placedinto the respective well of a 2.2 ml 96-deepwell plate (VWR 732-0585).Two steel balls (5 mm diameter, KGM Kugelfabrik GmbH, part No. 1.3541)were placed into each well and 500 μl homogenization buffer (videinfra), 20 μl DTT (1 M, Sigma 43816), 50 μl of proteinase K solution(Qiagen, 19133) was added. Furthermore, 10 μl working solution analyteand 10 μl working solution internal standard was added into each well ofthe standards to give the corresponding final concentrations of (5, 10,15, 20, 50, 75, 90, 100 μg/ml; 25 μg/ml IS (Internal Standard)).Standard 0 and recovered material were spiked with 10 μl of workingsolution internal standards only; recoveries were spiked with 10 μl ofworking solution analyte after the entire extraction process and priorto analysis.

Samples were processed as follows. A piece of approximately 50 mg oftissue was cut from the respective organ tissue, weighed and placed intothe respective well of a 96-deepwell plate. Two steel balls were placedinto each well and 500 μl homogenization buffer 20 μl DTT (1 M), 50 μlof proteinase K solution was added. The plate was sealed with STAR labfoils (StarLab E 2796 3070) and samples were homogenized using a QiagenTissue Lyzer 3×30 s at 17 Hz. Subsequently, the plate was incubated in awater bath for 2 hours at 55° C. followed by transfer of the samples toa new 96-deepwell plate using an automated liquid-handling system(TomTec Quadra 3). After the addition of 200 μl ammonium hydroxide (25%)and 500 μl phenol/chloroform/isoamyl alcohol (25:24:1) the plate wasvortexed using a Multitubevortex for 5 min. Subsequently, the plate wasincubated for 10 min at 4° C. and centrifuged at 4° C. for another 10min at 3500 RCF. The plate was then passed to the TomTec system whichwas used to remove the aqueous layer. The remaining organic layer waswashed by adding 500 μl water. The aqueous phase was again removed usingthe TomTec system. The aqueous phases were combined, 50 μl HCl (1 N),500 μl SAX Load High buffer (see below) and 300 μl acetonitrile wereadded, and the resulting solution was mixed thoroughly by up-and-downpipetting using the TomTec system. The program “SPE extraction of tissuesamples 100416” was used for the subsequent solid-phase extractionprocedure.

VARIAN Bond Elut 96 square-well SAX 100 mg (Cat. No.: A396081C) wereequilibrated with acetonitrile, water and SAX load buffer (see below),samples were loaded and washed with SAX load buffer. The samples wereeluted with SAX elute buffer (vide infra) and subsequently diluted withSAX/RP dilution buffer (vide infra). WATERS Oasis HLB LP 96-well Plate60 μm 60 mg (Part No. 186000679) were equilibrated with acetonitrile,water and SAX dilution buffer (see below). The samples were loaded andthe cartridge washed with water. The samples were eluted with RP elutebuffer (vide infra). Freeze the elution plate for 1 hour at −80° C. andlyophilize to dryness. The dried samples were reconstituted in 50 μlwater and dialyzed for 60 min against water using Thermo Slide-A-Lyzer.The samples were then subjected to CGE analysis on a Beckman CoulterPACE/MDQ system. The conditions were: (i) Capillary: eCAP DNA, neutral,21 cm, 100 μm I.D. (Beckman #477477); (ii) Capillary temperature: 20°C.; (iii) Sample storage temperature: 10° C., (iv) Gel: ssDNA 100 R(Beckman #477621) (v) Buffer: Tris/boric acid/EDTA buffer containing 7 MUrea (Beckman #338481) (vi) Detection wavelength: 260 nm; (vii)Separation voltage: 30 kV; (viii) Injection time: 60 s; (ix) Injectionvoltage: 10 kV; (x) Run time: 20 minutes; (xi) Data acquisition rate: 4pt/sec. Analysis was done using the Karat 7.0 software (Beckman).

In vitro dimer stability in murine and monkey plasmas and liverhomogenates was assessed using the assay described above. Subsequentlyto the incubation, samples were extracted with the phenol/chloroformextraction method and analyzed by LC-MS, as described above. FIG. 2illustrates in vitro dimer stability in murine or monkey plasmas anddegradation of dimer in liver homogenates as determined by LC-MS. FIGS.2A and 2B demonstrate slow degradation of both ApoC3 ASO monomer (SEQ IDNO: 1, designated as per Example 2(E)) and cleavable ApoC3-ApoC3 ASOdimers (SEQ ID NO:2 and SEQ ID NO:4) in murine and monkey plasmasrespectively. FIG. 2C demonstrates efficient degradation of thecleavable ApoC3-ApoC3 ASO dimers (SEQ ID NO:2 and SEQ ID NO:4) and therelative stability ApoC3 ASO monomer (SEQ ID NO: 1) in mouse liverhomogenate. FIG. 2D shows cleavable SEQ ID NO: 18) and noncleavable SEQID NO: 19) ApoB-ApoB ASO homodimers incubated in murine plasma or liverhomogenate, demonstrating stability of both types of molecules inplasma, and a more efficient degradation of the cleavable version in theliver homogenate.

Example 4: In Vitro Tests of Various Linker Designs with ApoC3 ASOHomodimers (FIG. 3A) Cell Culture Protocol

Human hepatocarcinoma cells (Hep3B) were acquired from the “DeutscheSammlung von Mirkoorganismen und Zellkulturen GmbH” (DSMZ). For the KDstudies, 3.000-10.000 cells/well were seeded (1-3 days prior totreatment) into 96 multi-titer plates yielding 70-80% confluence on theday of treatment. For assays using lipotransfection delivery techniques,cells were incubated with indicated concentrations of ASO formulatedwith 0.3 μl Lipofectamine 2000 (L2k) for 48 hr in Earle's Balanced SaltSolution (Lonza, Verviers, Belgium) with L-glutamine (2 mM).

Knock-Down Analysis Protocol

Following the treatment period mRNA levels of target and reference (ahousekeeping gene) mRNA was determined by the Quanti Gene Assay(Affymetrix, Santa Clara, Calif., USA) according to the manufacturesstandard protocol. Prior to lysis, cell viability was analyzed by CellTiter Blue Assay (Promega, Madison, Wis., USA). Inactive, scrambled, ASOwas used as negative control and reference (SEQ ID NO:31). TheQuantiGene 2.0 assay (Affymetrix, Santa Clara, Calif.) was utilized tomeasure the expression level of target genes in Hep3B cells before andafter the incubation with the ASOs. Human ApoB/ApoC3 probes andhousekeeping gene PPIB probes were purchased from Affymetrix. Standardassay procedures were carried out according to the manufacturer'srecommendations. On the day of harvesting, 200 μl/well of lysis buffer(with 1:100 protease K) was added to the cells. A total of 60 μl oflysate was used for human ApoC3 probes, while 20 μl lysate was used forhuman ApoB and PPIB probes respectively. Assay plates were read on theGloRunner Microplate Luminometer (Promega Corp, Sunnyvale, Calif.). Thedata were normalized against housekeeping gene PPIB.

Transfection Protocol

Hep3B cells were treated with 8 consecutive concentrations (0.001,0.006, 0.03, 0.2, 0.8, 4.0, 20 and 100 nM) of oligonucleotide wereformulated with the Lipotransfection agent. mRNA content and cellviability were determined after 48 hr of treatment.

The results of the above experiments are presented in FIG. 3A. Allhomodimers derived from the human sequence show knockdown. Homodimerswith thiol (S—S) bridges (SEQ ID NOs:2 and 4) showed increasedcytotoxicity. At the same time, the homodimer made from the murine ApoC3ortholog (SEQ ID NO:6) was ineffective

Example 5: In Vitro Comparisons of Cleavable Vs. Noncleavable LinkerDesigns with ApoC3 Homodimers (FIGS. 3B, 3C, 3J, 3K)

Cell were treated and analyzed as described in Example 4. For “gymnoticdelivery,” the cells were not transfected with the ASO, but instead wereincubated with indicated concentrations of unformulated ASO in MEM withhigh glucose (6 g/l; Invitrogen, Carlsbad, Calif., USA) withoutL-glutamine for 8 days. The results are presented in FIGS. 3B, 3C, 3Jand 3K.

When using lipotransfection techniques, the ApoC3 homodimers with moreeasily cleavable linkers (FIG. 3B, SEQ ID NOs: 15 and 17) showed ahigher knock-down activity than their less cleavable counterpart (FIG.3B, SEQ ID NO: 16). The same effect was seen with gymnotic delivery(FIG. 3C). FIG. 3J shows that the knock-down activity from the ApoC3homodimer (SEQ ID NO: 15) is better compared to the same sequence usedas monomer (SEQ ID NO:30). FIG. 3K shows that the ApoC3 homodimer, ifconnected via a metabolically unstable linker (SEQ ID NO: 15), is muchmore effective than its counterpart connected by a stable linker (SEQ IDNO: 16).

Example 6: In Vitro Tests of Cleavable Vs. Noncleavable Linker Designswith ApoB Homodimers (FIGS. 3D, 3E, 3H, 3I)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The results are presented in FIGS.3D, 3E, 3H and 3I. In lipotransfection assays, the ApoB homodimers witheasily cleavable linkers (FIG. 3D, SEQ ID NOs: 18, 20) showed a higherknock-down activity than their metabolic more stable analog (FIG. 3D,SEQ ID NO: 19). The same effect was seen with gymnotic delivery (FIG.3E). FIG. 3H shows that the knock-down activity from the ApoB homodimer(SEQ ID NO:18) is better compared to the same sequence used as a monomer(SEQ ID NO:13). FIG. 3I shows that the ApoB homodimer, if connected viaa metabolically unstable linker (SEQ ID NO:18), is much more effectivethan its counterpart connected by a stable linker (SEQ ID NO:19).

Example 7: In Vitro Tests of Cleavable Linkers of Different Lengths withApoB Homodimers (FIG. 3F, 3G)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The results are presented in FIGS.3F and 3G. For FIG. 3F, increasing numbers of DNA-phosphodiesterlinkages (ranging from one (SEQ ID NO:44) to eight (SEQ ID NO:49)) wereused to link the ApoB ASO sequences. The increasing the length of thelinker did not have a significant effect on the knockdown activity ofthe homodimer. FIG. 3G demonstrates that using RNA-phosphorothioatelinkers of different lengths (from one (SEQ ID NO:51) to four (SEQ IDNO:54)) also did not produce a significant impact on the knockdownactivity of the homodimer.

Example 8: In Vitro Activity Assessment of Knock-Down Activity ofCleavable ApoB/ApoC3 ASO Heterodimers Using Lipotransfection andGymnotic Delivery (FIGS. 4A and 4B)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The results are presented in FIGS.4A and 4B, wherein the monomers for ApoC3 (SEQ ID NO:30) and ApoB (SEQID NO: 13) show specific knock-down of the target mRNA, the ApoC3/ApoBheterodimers (SEQ ID NOs:21 and 22) show an intrinsic knock-downpotential for both targets, independent of the transfection method used(FIG. 4A—lipotransfection; FIG. 4B—gymnotic delivery).

Example 9: In Vitro Activity Assessment by Gymnotic Delivery forKnock-Down Activity of Cleavable ApoB/ApoC3 Heterodimers with VariousChemical Modifications (FIGS. 4C, 4D, 4E, 4F, 4G, 4H, 4I, and 4J)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The results are presented in FIGS.4C and 4D. In FIG. 4C, all ApoC3/ApoB heterodimers with differentmodifications (e.g., 2′-OMe, 2′F, 5-Prop.) showed a comparableknock-down activity toward both targets. FIG. 4D shows that also5-propynyl modifications (SEQ ID NOs:41 and 42) and different amounts ofLNA motifs (SEQ ID NO:40) do not change the overall knock-down activity.However, using a G-clamp modification for the ApoB ASO sequence (SEQ IDNO:38) decreases the knock-down potential for ApoB mRNA. FIGS. 4F-Jdepict the individual heterodimers versus the monomers used for thedesign. In FIG. 4E, the heterodimer (SEQ ID NO:33) assembled from SEQ IDNO:13 and SEQ ID NO:55 increases in knock-down activity toward bothtargets. In FIG. 4F, the heterodimer (SEQ ID NO:34) assembled from SEQID NO:13 and SEQ ID NO:56 increased in potency in lower concentrationonly for the ApoB target. In FIG. 4G, the heterodimer (SEQ ID NO:35)assembled from SEQ ID NO:13 and SEQ ID NO:57 increased in potency inlower concentrations for ApoB, while losing activity for ApoC3. In FIG.4H, the heterodimer (SEQ ID NO:36) assembled from SEQ ID NO:13 and SEQID NO:58 increased in knock-down potency in lower concentrations forApoB, while losing activity for ApoC3. In FIG. 4I, the heterodimer (SEQID NO:39) assembled from SEQ ID NO:13 and SEQ ID NO:1 increased inpotency in lower concentrations for ApoB, while showing a strongincrease in knock-down activity for ApoC3. In FIG. 4J, the heterodimerSEQ ID NO:21 assembled from SEQ ID NO: 13 and SEQ ID NO:30 showed nomodification of knock-down for ApoB, while ApoC3 knock-down activitydecreased.

Example 10: Direct Comparison of Knock-Down Activity of a CleavableHif-1Alpha/Survivin Heterodimer Versus its Parent Monomers UsingGymnotic Delivery (FIG. 4K)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The diagram in FIG. 4K depicts thatthe assembled HiF-1a/Survivin heterodimer (SEQ ID NO:23) inherits theindividual knock-down potentials of both parent sequences (SEQ ID NOs:27and 28).

Example 11: Direct Comparison of Knock-Down Activity of a CleavableHIF-1Alpha/ApoB Heterodimer Versus its Parent Monomers Using GymnoticDelivery (FIG. 4L)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The diagram in FIG. 4L depicts thatthe assembled HIF-1alpha/ApoB heterodimer (SEQ ID NO:25) inherits theindividual knock-down potentials of both parent sequences (SEQ ID NOs:13and 27).

Example 12: Direct Comparison Knock-Down Activity of a CleavableHIF-1Alpha/ApoB/ApoC3 Heterotrimers Versus its Parent Monomers by UsingGymnotic Delivery (FIG. 4M)

Cell culture, knock-down analysis and transfection procedures wereperformed as described in Example 5. The diagram in FIG. 4M depicts thatthe assembled HIF-1alpha/ApoB/ApoC3 heterotrimer (SEQ ID NO:26) inheritsthe individual knock-down potentials of all parent sequences (SEQ ID NO:13, SEQ ID NO:27, SEQ ID NO:30). A decrease in activity was observed forApoC3 and ApoB.

Example 13: Comparison of Dimer and Monomer Activity In Vivo

Acute in vivo activity assessments were performed in male and femalehuman ApoC3 transgenic mice (Jackson Labs Stock 905918, B6; CBA Tg(APOC3) 3707Bres/J), which are on a C57BL/6 background and express thehuman apoC3 gene including the human promoter. Male (22-30 g) and femalemice (20-25 g) employed in this study were 10 weeks old and fed regularchow diet.

ApoC3 ASO homodimers (SEQ ID NOs:4, 5, 2, or 3) or ApoC3 ASO monomer SEQID NO: 1 were formulated in sterile PBS pH7.0 (Gibco) for each doseimmediately before subcutaneous (sc) injection. Animals wereadministered equal volumes (100 μl) of the homodimers or monomer via scroute between the shoulder blades. A control group was treated usingequal volumes of PBS in parallel. Each treatment group consisted of 3male and 4 female transgenic mice.

Mouse blood was collected at Day 0 and Day 7 via submandibular puncture(50-75 μl), as well as at study termination (Day 14) by cardiacpuncture, post-euthanasia. Blood was collected in serum separator tubesat room temperature and allowed to clot for 30 minutes. Tubes were spunat 1000 rpm for 5 min at room temperature and serum above separatorlayer was collected and immediately aliquotted and frozen at −80° C. forfuture analysis. ApoC3 protein was determined using an ELISA (Wang etal., J. Lipid Res., 2011, 52(6):1265-71).

Effects on ApoC3 expression in the liver were also assessed at studytermination (Day 14) and baseline ApoC3 mRNA levels were determined froma group of mice euthanized on Day 0 of the study. Liver lobes wereexcised immediately after euthanasia and snap frozen in liquid nitrogen.RNA was subsequently isolated and ApoC3 mRNA expression was determinedusing the Affymetrix bDNA kit (QuantiGene, Affymetrix). The ApoC3 mRNAexpression was normalized to mouse GAPDH, a housekeeper gene, andreported as percent ApoC3 knockdown (KD) when compared to a PBS-treatedcontrol group.

The results of the in vivo studies are shown in FIGS. 5A-C, whichdemonstrate that under the conditions tested, the time course ofknock-down depended on the type of linker used to connect the twoantisense moieties in the dimeric antisense ODN. FIG. 5A demonstrates anassociated increased reduction of liver ApoC3 mRNA levels in human ApoC3transgenic mice following treatment with the endonuclease-sensitivephosphodiester-linked homodimers (SEQ ID NO:4 and SEQ ID NO:2). HumanApoC3 transgenic mice were administered a single subcutaneous dose ofhomodimers SEQ ID NO:5 or 3, which are disulphide-linked homodimers ofthe same monomer (each at 10 mg/kg), or vehicle. SEQ ID NO:4 and 2exhibited an increased reduction of liver ApoC3 mRNA levels compared tothe monomer (SEQ ID NO: 1) after 14 days. FIGS. 5B and 5C show ApoC3protein knock-down 7 days (FIG. 5B) and 14 days (FIG. 5C) after a single10 mg/kg dose of the monomer and dimeric LNA gapmers (SEQ ID NO:4 and 3)in human ApoC3 transgenic mice. The 3′3′-phosphodiester-linked dimerwith a total of eight phosphodiester linkages (SEQ ID NO:4) shows thefastest onset of knockdown after a single 10 mg/kg dose. Thisdemonstrates that the pharmacokinetic/pharmacodynamic properties can bemodulated by selecting a desirable linker.

Example 14: Biodistribution of Dimers

In a separate in vivo experiment, the bio-distribution of three dimersSEQ ID NOs:4, 2 and 3 was investigated in mice. The cleavage productswere analyzed by capillary gel electrophoresis (CGE) which was performedon a PACE/MDQ system (Beckman Coulter) equipped with the Karat 7.0software (Beckman Coulter). Further parts were: eCAP DNA capillary,neutral, 21 cm, 100 μm I.D. (Beckman #477477); ssDNA 100 R gel (Beckman#477621); buffer: Tris/boric acid/EDTA buffer containing 7 M urea(Beckman #338481). The cleavage products were further characterizedusing LC-ESI-TOF experiments which were performed on a Bruker Esquire6000 and an Agilent 1200 HPLC system, together with Waters ACQUITY UPLCOST C18 1.7 μm (part #186003949) column. Tissue homogenization was donewith a Multi-Tube Vortexer (VWR) and Lysing Matrix D (MP Biomedicals).Plate shaking was done using a VarioMag monoshaker. Deep-well plateswere from VWR (2.2 ml, cat. No. 732-0585) and were sealed with Clearseal diamond foil (Thermo, cat. No. 732-4890) prior to tissuehomogenization and were resealed for phenol/chloroform-extraction usingRe-Seal (3M Empore 98-0604-0472-4 adhesive). Acetonitrile was purchasedfrom Merck. Phenol/chloroform/isoamyl alcohol (25:24:1, P2069-100ML) anddithiothreitol (DTT, cat. No. 43816) were from Sigma, Proteinase K wasfrom Qiagen (cat. No. 19133), Slide-A-lyzer (200 μl, 10 kDa cut-off)were purchased from Fisher Scientific. High-grade 18 MOhm⁻¹ water(Millipore Milli-Q system) was used for reagent and sample preparations.A TomTec Quadra3 system was used for all liquid handling steps.

Plasma and Liver Homogenate Stability Experiments

Stock solutions with a final concentration of 600 μM and a final volumeof 100 μL have been prepared of all oligonucleotides.

Twelve pieces of approximately 50 mg of liver from CD1 mouse (female,Charles River) were added to individual Lysing matrix tubes. Acalculated volume of 1×PBS to give a final concentration of 5% liver(W/W) was added to each of the twelve tubes. All samples werehomogenized using a Biorad Fast prep System. The resulting homogenatesolutions were combined to give about 12 ml of 5% liver homogenate in1×PBS which was subsequently used for incubation.

Plasma was Na-Citrate plasma from female NMRI mouse (Charles River)K-EDTA plasma from male Cynomolgous monkey (Seralab International).

Four samples of each oligo were prepared representing each individualincubation time point (0, 7, 24 and 48 h) in mouse and monkey plasma andin mouse liver homogenate, respectively. In addition, a blank sample anda recovery sample were prepared of each oligo and incubation matrix.Generally, plasma samples were prepared by adding 5 μl of the 600 μMoligo stock solution to 95 μl of mouse or monkey plasma, respectively,with a final oligo concentration of 30 μM. Recovery samples wereprepared by adding 5 μl of water to 95 μl of plasma. Blank samples areoligo in water with a final concentration of 100 μM. Liver samples andrecoveries were prepared equally; apart from the fact that liverhomogenate in PBS was used instead of plasma.

Analysis of the Study Samples

Samples were analyzed in 96-well plate format. A standard curve with 8standards (5, 10, 15, 20, 50, 75, 90, 100 μg/ml; 25 μg/ml IS), astandard 0 (0 μg/ml; 25 μg/ml IS) and three recovery samples (20, 50,100 μg/ml; 25 μg/ml IS) has been prepared for each oligo. Samples andstandards of one particular oligo were analyzed together on the sameplate.

Standards were prepared as follows. A piece of approximately 50 mg oftissue was cut from the respective organ tissue, weighted and placedinto the respective well of a 2.2 ml 96-deepwell plate (VWR 732-0585).Two steel balls (5 mm diameter, KGM Kugelfabrik GmbH, part #1.3541) wereplaced into each well and 500 μl homogenization buffer (vide infra), 20μl DTT (1 M, Sigma 43816), 50 μl of proteinase K solution (Qiagen,19133) was added. Furthermore, 10 μl working solution analyte and 10 μlworking solution internal standard was added into each well of thestandards to give the corresponding final concentrations of (5, 10, 15,20, 50, 75, 90, 100 μg/ml; 25 μg/ml IS). Standard 0 and recoveries werespiked with 10 μl working solution internal standards only; recoverieswere spiked with 10 μl working solution analyte after the entireextraction process and prior to analysis.

A piece of approximately 50 mg of tissue was cut from the respectiveorgan tissue, weighted and placed into the respective well of a96-deepwell plate. Two steel balls were placed into each well and 500 μlhomogenization buffer 20 μl DTT (1 M), 50 μl of proteinase K solutionwas added. The plate was sealed with STAR lab foils (StarLab E 27963070) and samples are homogenized using a Qiagen Tissue Lyzer for 3×30 sat 17 Hz. Subsequently the plate was incubated in a water bath for 2 hat 55° C. followed by transfer of the samples to a new 96-deepwell plateusing an automated liquid-handling system (TomTec Quadra 3). After theaddition of 200 μl ammonium hydroxide (25%) and 500 μlPhenol/Chloroform/Isoamyl alcohol (25:24:1) the plate was vortexed usinga Multitubevortex for 5 min. Subsequently, the plate was incubated for10 min at 4° C. and centrifuged at 4° C. for another 10 min at 3500 RCF.The plate was then handled to the TomTec System which was used to removethe aqueous layer. The remaining organic layer was washed by adding 500μl water. The aqueous phase was again removed using the TomTec system.The aqueous phases were combined, 50 μl HCl (1 N), 500 al SAX Load Highbuffer (vide infra) and 300 μl acetonitrile was added and the resultingsolution was mixed thoroughly by up and down pipetting using the TomTecsystem. (‘The program “SPE extraction of tissue samples 100416” was usedfor the subsequent solid-phase extraction procedure).

Briefly: VARIAN Bond Elute 96 square-well SAX 100 mg (Cat. No. A396081C)were equilibrated with acetonitrile, water and SAX load buffer (seebelow), samples were load and washed with SAX load buffer. The sampleswere eluted with SAX elute buffer (vide infra) and subsequently dilutedwith SAX/RP dilution buffer (vide infra). WATERS Oasis HLB LP 96-wellPlate 60 μm 60 mg (Part No.: 186000679) were equilibrated withacetonitrile, water and SAX dilution buffer (vide infra). The sampleswere load and the cartridge washed with water. The samples were elutedwith RP elute buffer (vide infra).

Freeze the elution plate for 1 h at −80° C. and lyophilize to dryness.The dried samples are reconstituted in 50 μl water and dialyzed for 60min against water using Thermo Slide-A-Lyzer. The samples were thensubjected to CGE analysis on a Beckman Coulter PACE/MDQ system. Theconditions were: (i) Capillary: eCAP DNA, neutral, 21 cm, 100 μm I.D.(Beckman #477477); (ii) Capillary temperature: 20° C.; (iii) Samplestorage temperature: 10° C., (iv) Gel: ssDNA 100 R (Beckman #477621) (v)Buffer: Tris/boric acid/EDTA buffer containing 7 M Urea (Beckman#338481) (vi) Detection wavelength: 260 nm; (vii) Separation voltage: 30kV; (viii) Injection time: 60 s; (ix) Injection voltage: 10 kV; (x) Runtime: 20 minutes; (xi) Data acquisition rate: 4 pt/sec. Analysis wasdone using the Karat 7.0 software (Beckman).

All samples and recoveries were incubated at 37° C. A sample of eacholigo and type of matrix was cooled to room temperature after 0, 7, 24and 48 h and was subjected to Phenol/Chloroform purification. To thisend, 370 μl of ammonium hydroxide (15%), 10 μl dithiothreitole (DTT, 1M, Sigma 43816) and 800 μl premixed Phenol/Chloroform/Isoamyl alcohol(Sigma P2069) was added to each sample. The sample was vortexed for 10min at maximum vortex speed and then incubated at 4° C. for 20 min.Subsequently, the sample was centrifuged at 3500 RFC for 20 min at 4° C.and 400 μl of the aqueous layer were removed and dried in a lyophilizer.The dried samples were dissolved in water (100 μl). The recovery sampleswere dissolved in water (95 al) and spiked with 5 μl of the respectiveoligo stock solution (600 μM). Samples were analyzed by LC-MS (Agilent1200, Bruker Esquire 3000) using a Waters Acquity UPLC OST C18 column(1.7 μm, 2.1×50) with HFIP/TEA/water (385 mM1,1,1,3,3,3-hexafluoroisopropanol, 14.4 mM triethylamine in water) asbuffer A and methanol as buffer B and a flow rate of 0.3 ml/min at acolumn temperature of 60° C. The following gradient was used: 3 min at5% B, 5-15% B in 2.5 min (10%/min), 15-23% B in 5.5 min, 23-30% B in 3min, 30-100% B in 0.5 min, 5 min at 100% B, 100-5% B in 0.5 min, 5 minat 5% B.

Surprisingly, the levels of dimers in the liver (organ target for ApoBand ApoC3) and kidney were dramatically increased after a single i.v.bolus injection. It was found that about 10 to 16% monomeric metaboliteof the total dose in liver 24 hours after injection of the dimers (Table5), while previously it was known that only 2 to 5% of the total dose ofthe monomeric 14-mer (SEQ ID NO: 1)) in mice or monkeys (Table 6) wasdetected in a separate study. Accordingly, dimers exhibitedsignificantly higher biodistribution to liver and kidney as compared tothe monomers. Table 5 shows organ-distribution of antisense dimers SEQID NO:2, 3 and 4 as percent of total administered dose 24 hrs after asingle i.v. bolus injection into mice. (Peak 1 refers to the degradationproduct, whereas Peak 2 is remaining dimer starting material. The sum ofboth components represents the percentage of total dose in thecorresponding organ.) Organ-distribution of monomeric SEQ ID NO: 1 aspercent of total administered in mice and monkeys in previous studies ascompared to the dimers (last row) is shown Table 6. Percent total dosecalculation based on: 5 kg monkey, 135 g liver, 30 g kidney (Davies etal., Pharm. Res., 1993, 10 (7):1093).

TABLE 5 Linker Oligo Animal Peak 1 Peak 2 % totaldose* Liver Diester SEQID NO: 2 1-3  8.1 μg/g  9.1 μg/g 14% SS SEQ ID NO: 3 4 & 6 20.8 μg/g —16% Diester SEQ ID NO: 4 7-9 12.2 μg/g — 10% doubler Kidney Diester SEQID NO: 2 1-3 16.7 μg/g 58.1 μg/g 15% SS SEQ ID NO: 3 4-6 29.9 μg/g 47.4μg/g 15% Diester SEQ ID NO: 4 7 & 9 54.9 μg/g  6.4 μg/g 12% doubler*based on 25 g mouse, 2 g liver, 0.5 g kidney

TABLE 6 Study Liver Kidney SEQ ID NO: 1  2.5-5%  1.2-3% Monkey tox  13μg/g (2 mpk)  60 μg/g (2 mpk) 2, 10, 60 mpk  50 μg/g/10 mpk) 300 μg/g(10 mpk) Necrop @ day 25 300 μg/g (60 mpk) 800 μg/g (60 mpk) 4 doses @day 1, 8, 15, 22 SEQ ID NO: 1 0.3% total dose 0.4% total dose Mouse tox 3.7 μg/g  21 μg/g Twice/week 25 mpk Necrop @ day 15 (100 mpk) SEQ IDNO: 1 4.1% (30 mpk)   6% (30 mpk) Monkey PK 3.6% (3 mpk)  16% (3 mpk)Single bolus iv  45 μg/g (30 mpk) 300 μg/g (30 mpk) 3, 30 mpk  4 μg/g (3mpk)  80 μg/g (3 mpk) Necrop @ 24 h SEQ ID NO: 2, 3 or 4  10-16%  12-15%(dimers)  16 μg/g (10 mpk)*  70 μg/g (10 mpk)* Mouse Single bolus iv*mean over 3 *mean over 3 10 mpk Necrop @t 48 h

The high levels of the monomeric equivalent (peak 1) were verysurprising, since most of the injected dimer was already processed to amonomeric form (left peak 1 with shortest retention time, as shown inFIG. 6). In the case of dimer of SEQ ID NO:2, the intact dimer wasdetected at 6.458 min), as well as the monomer and the monomer with anadditional dT (SEQ ID NO: 1 plus dT) from the incomplete cleavage of thelinker. The internal standard (IS) is poly-(dT)₃₀ phosphorothioate. Thedimers SEQ ID NOs:4 and 2 were already completely converted to themonomeric forms comprising the monomer (SEQ ID NO: 1) and the monomerplus dT. In case of dimer SEQ ID NO:3 with a disulfide linker, themonomeric cleavage product was slightly larger than monomer resultingfrom reductive disulfide cleavage and is indicated as “#1 plus X” in thefigure, where X is a yet unidentified organic radical with the molecularweight of less 100 Da. It could be hypothesized that that “#1 plus X”results from oxidative cleavage rather than reductive cleavage of thedisulfide bond. If the dimers had been already cleaved in the serum, thebio-distribution to liver and kidneys should not have increased sodramatically as compared to monomers. Thus, the stability of the dimersin plasma and in liver homogenates was investigated. It was demonstratedin Example 3 that plasma stability of the dimers is relatively high over48 hours, while the dimers are rapidly cleaved in liver homogenates.Further cleavage product analysis of samples extracted from the liverhomogenate treatment showed that the dimer is completely converted tothe monomeric form. This observation is compatible with abio-distribution mechanism, in which dimers are relatively stable afterinjection into animal. The dimers distributed more efficiently to theorgans like liver and kidney as opposed to the corresponding monomer. Inthe organs (e.g., liver and kidney), the dimer is cleaved to the monomerand can act as a normal antisense oligonucleotide. Since the dimers arestable in serum (plasma), the linkers can be designed to undergo anorgan-specific cleavage by using appropriate linker chemistry.

Example 15: In Vivo Activity Assessment of a Cleavable ApoC3/ApoB ASOHeterodimer

In vivo activity of a heterodimer of a human ApoC3 ASO linked to an ApoBASO with a cleavable linker was assessed in male and female human ApoC3transgenic mice which were 14-18 weeks old at termination.

The ApoC3/ApoB ASO heterodimer (SEQ ID NO: 21) or a non-targeting ASO(SEQ ID NO: 119) were formulated in sterile saline (pH7.0) immediatelybefore intravenous (iv) injection via the tail vein. Animals wereadministered heterodimer (0.3, 1, 3, or 10 mg/kg) or negative controlASO (10 mg/kg) or saline (0 mg/kg) as a vehicle control in a volume of 5ml/kg.

Groups of mice consisted of 2 male and 2 female transgenic mice whichwere terminated on days 1, 3, 7, 14 and 29 after treatmentadministration. After euthanasia by CO₂ inhalation, blood was obtainedby cardiac puncture (0.5-1 ml). Livers were dissected, weighed, and afragment saved in a labeled histology cassette snap frozen by immersionin liquid nitrogen. Liver samples were maintained at −80° C. forsubsequent analyses.

Each blood sample was divided in half. Serum was prepared in serumseparator tubes which were allowed to clot for 4 hours on ice. Plasmawas prepared in EDTA-containing tubes which were maintained on ice untilprocessed. Tubes were spun at 10,000 rpm for 5 min at 4° C. andsupernatants collected and frozen at −80° C. for future analyses.

Quantification of Target mRNAs

Total liver RNA was isolated in TRIzol reagent (Ambion) from snap frozentissue homogenized in Fastprep24 Lysing Matrix D tubes (MP Biomedicals).Trizol-chloroform extraction was followed by further purification usinga column-based method (Qiagen, RNeasy) as per manufacturer'sinstruction. Purification included treatment with DNase I for 15 minutesat room temperature (Qiagen, Rnase-Free Dnase). RNA quantity and puritywere evaluated spectophotometrically by readings at 260 nm and 280 nm(Nanodrop). Liver fragments were lysed with RLT buffer and QIAshreddercolumns (Qiagen), and then purified by RNeasy columns as indicatedabove.

Samples were amplified as per manufacturer's instructions (Qiagen,Quantitect Probe RT-PCR kit). Quantitative real-time PCR (qRT-PCR) wasperformed in a 7900HT Fast Real-Time PCR System (Applied Biosystems).All samples were analyzed in triplicate in Microamp Optical 384 wellreaction plates (Applied Biosystems) and normalized with Gapdh signal asthe internal control. Primers were Apolipoprotein C-III (AppliedBiosystems, Mm00445670_m1 and Hs00163644_m1), Apolipoprotein B (AppliedBiosystems, Mm01545156_m1 and Hs01071209_m1), and Mouse GAPDH (AppliedBiosystems, 4352932E). Results are expressed as fold induction relativeto vehicle-treated samples.

Data for each of the target mRNAs were analyzed by two-way ANOVA using“time” and “treatment” as the variables in GraphPad Prism software.Bonferroni post-hoc tests were conducted when significant main effects(p<0.05) were observed.

The results of this in vivo experiment are shown in FIGS. 7A and 7B. Thedata demonstrate that SEQ ID NO: 21, an ApoC3/ApoB heterodimer ASO withan endonuclease sensitive phosphodiester linker, significantlydown-regulated liver expression of both target mRNAs [i.e, human APOC3(FIG. 7A) and mouse ApoB (FIG. 7B)]. Target mRNA knockdown was dependenton both administered dose and time. That is, in animals which receivedmore ASO construct, a greater target knockdown was observed. Thegreatest degree of knockdown for any dose level was observed during thefirst week post-administration, with significant effects persistinguntil 29 days post-administration, the longest time point at whichsamples were obtained.

Example 16: In Vivo Comparison of Heterodimer ASOs and Monomers: Effectson Target mRNAs

In vivo activity of three heterodimers of a human ApoC3 ASO linked to anApoB ASO, the ApoC3 ASO monomer, the ApoB ASO monomer and the physicalcombination of the two monomers was assessed in male human ApoC3transgenic mice which were 9-18 weeks old at termination.

An ApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable;SEQ ID NO: 21), or an ApoC3/ApoB ASO heterodimer linked with fourphosphothioate bases (stable; SEQ ID NO: 59), or an ApoC3/ApoB ASOheterodimer linked with PEG-6 (stable; SEQ ID NO: 60), or the ApoC3monomer ASO (SEQ ID NO: 30), or the ApoB monomer ASO (SEQ ID NO: 13), orthe physical combination of the ApoC3 and ApoB monomers (SEQ ID NO: 30plus SEQ ID NO: 13), or a non-targeting ASO (SEQ ID NO: 119) wereformulated in sterile SALINE (pH7.0) immediately before intravenous (iv)injection via the tail vein. Animals were administered equal molaramounts of heterodimer (0.3 Mol/kg˜3 mg/kg), monomer (0.3 μMol/kg˜1.3mg/kg), co-formulated monomers (0.3 μMol/kg each) or negative controlASO (0.3 μMol/kg˜1.4 mg/kg) or SALINE (0 mg/kg) as a vehicle control ata volume of 5 ml/kg.

Groups consisted of 6-7 male transgenic mice which were terminated 3 or14 days after treatment administration. After euthanasia by CO₂inhalation, blood was obtained by cardiac puncture (0.5-1 ml). Liverswere dissected, weighed, and a fragment put in a labeled histologycassette snap frozen by immersion in liquid nitrogen. Whole kidneys werealso stored in labeled histology cassettes and snap frozen in liquidnitrogen. Liver and kidney samples were maintained at −80° C. forsubsequent analyses.

Each blood sample was divided in half. Serum was prepared in serumseparator tubes which were allowed to clot for 4 hours on ice. Plasmawas prepared in EDTA-containing tubes which were maintained on ice untilprocessed. Tubes were spun at 10,000 rpm for 5 min at 4° C. andsupernatants collected and frozen at −80° C. for future analyses.

Data for each of the target mRNAs on either Day 3 or Day 14 wereanalyzed by one-way ANOVA followed by Dunnett's post-hoc test todetermine differences between treatments using GraphPad Prism software.

The effects of these treatments on in vivo target mRNAs in the liver areshown in FIGS. 8A and 8B. Data in these figures are plotted as %knockdown of the target mRNAs with knockdown of mouse apoB mRNA plottedon the x axis and knockdown of human ApoC3 (i.e., the transgene) plottedon the y axis. The data demonstrate that SEQ ID NO: 21, an ApoC3/ApoBheterodimer ASO with an endonuclease sensitive phosphodiester linker,was superior to all other treatments on both day 3 (FIG. 8A) and day 14(FIG. 8B) in the extent to which it down-regulated liver expression ofboth target mRNAs.

On day 3 (FIG. 8A), ApoB mRNA in the liver was significantly decreasedby all treatments, except the ApoC3-targeted ASO monomer (SEQ ID NO: 30)and the negative control ASO (SEQ ID NO: 119). In general, theeffectiveness of constructs given on day 0 to suppress target mRNAs wasweaker 14 days after treatment administration than observed 3 dayspost-treatment. Nevertheless, ApoB mRNA in the liver (FIG. 8B) wassuppressed by all treatments except the ApoC3-targeted ASO monomer (SEQID NO: 30), the ApoC3/ApoB ASO heterodimer linked with PEG-6 (stable;SEQ ID NO: 60, and the negative control ASO (SEQ ID NO: 119).Importantly, treatment with SEQ ID NO: 21, an ApoC3/ApoB heterodimer ASOwith an endonuclease sensitive phosphodiester linker resulted insignificantly greater knockdown of liver ApoB mRNA than any othertreatment at each of the times at which samples were taken (FIGS. 8A and8B).

Qualitatively similar results were observed for knockdown of human ApoC3mRNA in these human ApoC3 transgenic mice. On Day 3 (FIG. 8A), the ApoC3monomer (SEQ ID NO: 30), the physical combination of the ApoC3 and Apo Bmonomers (SEQ ID NO: 30 plus SEQ ID NO: 13), the ApoB monomer (SEQ IDNO: 13), and the ApoC3/ApoB ASO heterodimer linked with four diesterbases (cleavable; SEQ ID NO: 21) significantly decreased expression ofhuman ApoC3 mRNA. On Day 14 (FIG. 8B), only the ApoC3/ApoB ASOheterodimer linked with four diester bases (cleavable; SEQ ID NO: 21)significantly suppressed expression of human ApoC3. Similar to itseffectiveness in suppressing ApoB, administration of SEQ ID NO: 21resulted in significantly greater knockdown of liver human ApoC3 mRNAexpression than any other treatment (FIGS. 8A and 8B).

Example 17: Tissue Stability of Heterodimer ASOs

Hybridization assays were developed (see below) to measure the tissueconcentrations of the ApoC3/ApoB ASO heterodimer linked with fourdiester bases (cleavable; SEQ ID NO: 21), the ApoC3/ApoB ASO heterodimerlinked with four phosphothioate bases (stable; SEQ ID NO: 59), and theApoB monomer ASO (SEQ ID NO: 13) in plasma and homogenates of liver andkidney. Samples from the experiment described in Example 16 weremeasured.

Capture and Detection Probes:

Complementary hybridization probes to the hetero-dimeric ASOs weredesigned and custom synthesized with LNA-modified phosphodiesterbackbones (BioSpring GmbH). The capture probes contained an amino linker(C12-amino) and Spacer-18s (hexaethyleneglycole phosphate, PEG-282) atthe 5′-end. The detection probes contain Spacer-18s at the 3′-end of thespecific probe sequence and were biotin labeled at the 3′-end (Elfer etal., 2005). The specific sequences of capture and detection probes usedin the assays are showed in table below.

TABLE 7 Capture and Detection Probes used in Hybridization Assays SEQ IDProbe NO Sequences Dimer Capture 120 5′-(C12-amino)(Spacer-18) Probes(Spacer-18)-βG-βC-βA-βA- βA-βA-βA-βG-3′ Detection 1225′-βT-βC-βA-βG-βT-βG-βC- (Spacer-18)(Spacer-18)(dT-biotin)(biotin TEG)-3′ apoB Capture 124 5′-(C12-amino)(Spacer-18) ProbesSpacer 18)-βT-βG-βA-βA- βT-βA-βC-3′ Detection 121 5′-βC-βA-βA-βT-βG-βC-(Spacer-18)(Spacer-18)(dT- biotin)(biotin TEG)-3′

Chemistry Synthesis of Oligonucleotide

The procedure below covers the synthesis of two oligonucleotides [SEQ IDNO: 59(5-βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*T*dT*dT*dT*dT*βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-3′) and SEQ ID NO: 60(5′-βG*βZ*dA*dC*dT*dG*dA*dG*dA*dA*dT*dA*βZ*βT-HEG-βG*βZ*dA*dT*dT*dG*dG*dT*dA*dT*βT*βZ*βA-3′)].The synthesis was performed using a standard synthesis protocol on anAKTA oligopilot 10 Plus synthesizer using the conditions summarized inTable 8.

TABLE 8 Oligonucleotide Synthesis Conditions Column size/scale 3.5 ml/63μmol Solid support; loading Nittophase Universal Support; 100 μmol/gAmidite concentration 0.1M Amidite equivalents 4

The oligonucleotide was cleaved from solid support using a solution ofammonium hydroxide and ethanol (3:1) at 55° C. for 17 hours. The crudeoligonucleotides were purified in a two-step IEX-purification procedureusing a Source 30Q column and buffer system containing sodium hydroxide.The mass spectrometer analysis was done using ESI-MS and the purity wasestablished using HPLC and generic method. The endotoxin levels weremeasured using LAL-test procedure.

Synthesis of Capture and Detection Probes

This procedure covers the synthesis of both capture and detection probes[SEQ ID NO: 120(5′-(C12-amino)(Spacer-18)(Spacer-18)-βG-βC-βA-βA-βA-βA-βA-βG-3′); SEQID NO: 122 (5′-βT-βC-βA-βG-βT-βG-βC-(Spacer-18)(Spacer-18)(dT-biotin)(biotin TEG)-3′); SEQ ID NO: 123(5′-(C12-amino)(Spacer-18)(Spacer-18)-βT-βG-βA-βA-βT-βA-βC-3′) and SEQID NO: 121(5′-βC-βA-βA-βT-βG-βC-(Spacer-18)(Spacer-18)(dT-biotin)(biotinTEG)-3′)]. The synthesis was performed using a standard synthesisprotocol on an AKTA oligopilot 10 Plus synthesizer using the conditionssummarized in Table 9.

TABLE 9 Conditions Used to Synthesize Capture and Detection ProbesColumn size/scale 1.2 ml/22 μmol or 17 μmol Solid support; loadingNittophase Universal Support; 100 μmol/g Amidite concentration 0.1MAmidite equivalents for LNA 5 Amidite equivalents for Spacer-18 3 andNH2—Cl2-amino

The oligonucleotide was cleaved from solid support using a solution ofammonium hydroxide and ethanol (3:1) at 55° C. for 17 hours. The crudeoligonucleotides were purified in a two-step RP-/IEX-purificationprocedure. The RP-purification was by applying a TEAA-containing buffersystem, the IEX purification was carried out at physiologicalconditions. The mass spectrometer analysis was done using ESI-MS and thepurity was established using HPLC and generic method.

Tissue Sample Preparation:

Liver and kidney homogenate was prepared from animals treated withheterodimeric or monomeric ASOs. Tissue samples collected at specifiedtime points were minced and weighed in ready-to-use Lysing Matrix Dtubes containing 1.4 mm ceramic spheres beads (Catalogue#6913-100, MPBiomedicals). DNase/RNAse free water (Catalogue #SH30538.02, Thermo) wasadded to the tube with ratio of 5 or 10 mL per g of tissue. Each tissuesample was mixed and homogenized using a MP Biomedicals Fast Prep-24 at4° C. for 20 seconds twice. The tissue homogenate was stored in freezeror kept on ice before analyzed with the hybridization assay.

Preparation of Standards and Controls:

Standards and assay quality controls (QCs) were prepared in K2 EDTAplasma or control tissue matrix and diluted serially in 2-fold stepsfrom 100 ng/mL to 0.098 ng/mL. The QCs were set at 50 ng/mL, 40 ng/mL,10 ng/mL, 1 ng/mL and 0.4 ng/mL. The standards and QCs were analyzed bythe hybridization assay with the samples.

Hybridization Methods with Colorimetric Detection:

DNA-Bind plates (96-well) (Catalogue #2505, Costar) were coatedovernight at 4° C. with 100 μL of 50 nM capture probes in HEPES/1 mM Na₂EDTA buffer. The plates were then washed three times with wash buffer(Tris Buffer/0.1% Tween 20) and incubated in blocking buffer (PBS/3%BSA) for 1-2 hrs. 30 μL of Samples, Standards, and QCs were mixed with270 μL of 50 nM detection probe in hybridization buffer (4×SSC/0.5%Sarkosyl) in Costar cluster tubes and two 100 μL aliquots from themixture were transferred into 96-well PCR plate and denatured on thethermocycler for 12.5 minutes at 95° C. After the samples were cooled to40° C., they were transferred to DNA-Bind plate already coated withcapture probe. The plate was sealed and incubated at 40° C. for twohours. Following the hybridization, Poly-HRP Streptavidin conjugate(Catalogue # N200, Thermo) at 1:10,000 dilution in Poly-HRP dilutionbuffer (Catalogue # N500, Thermo) was added. Color development wasinitiated by adding SureBlue TMB substrate (Catalogue #52-00-00, KPL)and stopped with stop reagent for TMB substrate (Catalogue # S5814,Sigma).

Results:

The ApoC3/ApoB ASO heterodimer linked with four diester bases(cleavable; SEQ ID NO: 21) or the ApoC3/ApoB ASO heterodimer linked withfour phosphothioate bases (stable; SEQ ID NO: 59) were spiked into liveror kidney (n=2 each) and homogenized as described above. The homogenatewas divided into two aliquots. One of the aliquots was stored at −80°C., the other aliquot was placed at 37° C. for 15 hours before storageat −80° C. The two aliquots were thawed and analyzed together for theconcentration of heterodimeric ASOs and apoB monomer with thehybridization assay.

As shown in FIGS. 9A and 9B, concentrations of both heterodimers werelower after overnight incubation at 37° C., suggesting degradation intissue at physiological temperature. The ApoB monomer ASO was detectableas a metabolite in both liver and kidney samples spiked with theApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable;SEQ ID NO: 21) and the levels were more than 5 fold higher in samplesincubated at 37° C. After spiking with the ApoC3/ApoB ASO heterodimerlinked with four phosphothioate bases (stable; SEQ ID NO: 59), the ApoBmonomer ASO was only detectable in liver homogenates which had beenfrozen. Taken together, the data suggest that SEQ ID NO: 21 is degradedto active ApoB monomer (SEQ ID NO: 13) metabolite more readily from theApoC3/ApoB ASO heterodimer linked with four diester bases (SEQ ID NO:21) than from the ApoC3/ApoB ASO heterodimer linked with fourphosphothioate bases (stable; SEQ ID NO: 59).

Example 18: In Vivo Distribution of Heterodimer ASOs and ApoB MonomerASO

In plasma, heterodimer ASOs and the ApoB monomer were measured using themethods above in 2 pools of 3 individuals each after treatment with theApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable;SEQ ID NO: 21), the ApoC3/ApoB ASO heterodimer linked with fourphosphothioate bases (stable; SEQ ID NO: 59), the ApoB monomer ASO (SEQID NO: 13) or the physical combination of the ApoC3 and ApoB monomerASOs (SEQ ID NO: 30 plus SEQ ID NO: 13). As shown in FIG. 10, bothheterodimer ASOs were detected in plasma 3 days post-treatment. ApoBmonomer was also detected 3 days after treatment with the ApoB monomerASO alone or in physical combination with the ApoC3 monomer. However,ApoB monomer ASO was detected as a metabolite of the ApoC3/ApoB ASOheterodimer linked with four diester bases (cleavable; SEQ ID NO: 21) 3days after treatment, but not after administration of the ApoC3/ApoB ASOheterodimer linked with four phosphothioate bases (stable; SEQ ID NO:59), demonstrating that the endonuclease sensitive linker resulted inenhanced metabolism to active ASO monomers. None of the analytes weredetected in plasma pools taken 14 days after treatment.

Differences between heterodimer or monomer concentrations in tissueswere determined statistically by unpaired t-test (heterodimers) orone-way ANOVA followed by Bonferroni post-hoc comparisons (monomers)using GraphPad Prism.

In the kidney, measured concentrations of all administered constructsand the ApoB monomer metabolite decrease significantly between 3 and 14days after administration. The decline in the concentrations of theApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable;SEQ ID NO: 21) is the most rapid/marked, which is compatible with thehypothesis that the construct is most vulnerable to metabolism viacleavage of the linker (see FIGS. 11A and 11B). On both day 3 and day14, levels of the ApoB monomer are lowest after administration of theApoC3/ApoB ASO heterodimer linked with four phosphothioate bases(stable; SEQ ID NO: 59) while the levels of intact SEQ ID NO: 59 are thehighest of the constructs measured. Taken together, these observationsdemonstrate slower metabolism of the relatively stable phosphothioatelinker.

In liver, the ApoC3/ApoB ASO heterodimer linked with four diester bases(cleavable; SEQ ID NO: 21) was present at lower concentrations than theApoC3/ApoB ASO heterodimer linked with four phosphothioate bases(stable; SEQ ID NO: 59) after treatment with the respective constructs(see FIGS. 12A and 12B), demonstrating that SEQ ID NO: 21 is metabolizedmore quickly in liver. Monomer levels that were measured on day 3 and 14were significantly lower after administration of the ApoB monomer (SEQID NO: 13) either alone or in physical combination with the ApoC3monomer than after administration of with either of the measuredheterodimer of ApoC3/ApoB ASOs. On day 3, the concentration of ApoBmonomer present in the liver as a metabolite after heterodimeradministration was significantly higher after administration of theApoC3/ApoB ASO heterodimer linked with four diester bases (cleavable;SEQ ID NO: 21) than the ApoC3/ApoB ASO heterodimer linked with fourphosphothioate bases (stable; SEQ ID NO: 59), substantiating that thelinker designed for cleavage by endonucleases resulted in higherconcentration of active monomeric ASO metabolite within a few days ofadministration (see FIG. 12A). On day 14, the reverse was observed (seeFIG. 12B). The concentration of ApoB monomer present in the liver as ametabolite after heterodimer administration was significantly higherafter administration of the ApoC3/ApoB ASO heterodimer linked with fourphosphothioate bases (stable; SEQ ID NO: 59) than the ApoC3/ApoB ASOheterodimer linked with four diester bases (cleavable; SEQ ID NO: 21).Since the phosphothioate linked heterodimer also degrades in tissue,albeit at a slower rate, relatively more monomer is present at this timepoint. The levels of ApoB monomer, after its administration or as ametabolite of administered ASO heterodimers is related to target mRNAknockdown, e.g., the highest levels of ApoB monomer are present afteradministration of the ApoC3/ApoB ASO heterodimer linked with fourdiester bases (cleavable; SEQ ID NO: 21) and the highest level of targetmRNA knockdown is also observed in this treatment group (compare FIGS.12A and 12B to FIGS. 8A and 8B)

Example 19 Oligo Sequences

15-mer gapmer oligos were designed as single monomers or as homodimers(30-mers) linked by an oligo-dT linker (4 bases) via cleavablephosphodiester bonds. The oligos were designed to target either miR-122or MALAT-1 and consisted of three LNA-modified bases at each end of themonomer with 9 unmodified DNA bases in the center or gap region. Thegapmer design facilitated cleavage of the bound target mRNA by RNAseHresulting in a decrease in target mRNA (either miR-122 or MALAT-1). Thesequences of the following table correspond, from top to bottom, to SEQID NOS: 128 to 135.

Oligo ID Oligo Sequence 122gap-mono bCsbAsbTsTsGsTsCsAsCsAsCsTsbCsbCsbA122gap-dimer bCsbAsbTsTsGsTsCsAsCsAsCsTsbCsbCsbAoToToToTobCsbAsbTsTsGsTsCsAsCsAsCsT sbCsbCsbA 122gap-bTsbGsbAsAsGsGsTsTsCsCsTsCsbCsbTsbT control- mono 122gap-bTsbGsbAsAsGsGsTsTsCsCsTsCsbCsbTsbT control-oToToToTobTsbGsbAsAsGsGsTsTsCsCsTsC dimer sbCsbTsbT Malat1-bCsbTsbAsGsTsTsCsAsCsTsGsAsbAsbTsbG gap-mono Malat1-bCsbTsbAsGsTsTsCsAsCsTsGsAsbAsbTsbG gap-dimeroToToToTobCsbTsbAsGsTsTsCsAsCsTsGsA sbAsbTsbG Malat1-gap-bTsbTsbCsCsCsTsGsAsAsGsGsTsbTsbCsbC control-mono Malat1-gap-bTsbTsbCsCsCsTsGsAsAsGsGsTsbTsbCsbC control-dimeroToToToTobTsbTsbCsCsCsTsGsAsAsGsGsT sbTsbCsbC all bases are DNA b = LNAs = Phosphorothioate linkage o = Phosphodiester linkage

Animal Care and Treatments:

Animal experiments were conducted in an Association for the Assessmentand Accreditation of Laboratory Animal Care (AAALAC) facility under aconstant light-dark cycle, maintained on a standard mouse diet, andallowed ad libitum access to food and water. Mice were euthanized by CO₂inhalation. All mouse experiments were approved and conducted incompliance with the guidelines of the Institutional Animal Care and UseCommittee at Vivisource Laboratories, Inc. Female C57BL6/J mice wereobtained from the Jackson Laboratories (Bar Harbor, Me.) and femaleBalb/C mice obtained from the Charles River Laboratories.Oligonucleotides were dissolved in phosphate buffered saline (PBS) andadministered to mice based on body weight by subcutaneous injection.Mice were injected once per week (MALAT-1) at 50 mg/kg or twice per week(MIR-122) at 10 mg/kg or 50 mg/kg. Mice were sacrificed after one weekand at study termination (four weeks) and liver, kidney and plasmaharvested for further analysis.

Triglycerides, HDL and Total Cholesterol Measurements.

Blood was collected by cardiac puncture and total plasma harvested bycentrifugation in Minicollect tubes (Thermo Fisher). Plasmaconcentrations of Triglycerides, total cholesterol and LDL cholesterolwere determined by enzymatic assay (Bio Scientific) on a MolecularDevices SpectraMax M5 plate reader according to manufacturer'srecommendations.

RNA Extraction, Reverse Transcription and mRNA qPCR.

Tissue was disrupted using a FastPrep-24 tissue homogenizer (MPBio) andtotal RNA isolated using Trizol (Invitrogen) and miRNEasy columns(Qiagen). RNA concentration was assessed using RIboGreen plates(Molecular Probes) and a Molecular Dynamics M5 multimodal plate reader.250 ng of total RNA was reverse transcribed with random hexamers in a 50ml reaction using High Capacity Multiscribe Reverse Transcriptase. qPCRwas carried out using the equivalent of 12.5 ng cDNA in 20 μl reactionvolumes using MIR122 or MALAT-1 specific TaqMan primers and probes on aStep-One Plus thermocycler. Relative qPCR expression of individual geneswas normalized to the expression of reference genes GusB (accession#NM_010368), GAPDH (accession# NM_008084.2) or SNO-135 (accession#AF357323) RNA using the ΔΔCt method.

miR-122 Study Results

Two separate cohorts of C57Bl6/J mice (short and long dosing arm) wereanalyzed. The mice were females and, both cohorts were maintained onregular chow. Both cohorts were dosed at 10 mg/kg and 50 mg/kg bysubcutaneous injection twice a week (day 1 and 4). Targetingoligonucleotides used were 15 base gapmers with LNA at the ends (3-9-3)and full phosphorothioate linkage. Animals were euthanized at day 7(short arm) and day 28 (long arm). Dose Groups were n=5. The followingparameters were analyzed in an ex vivo analysis: ALT, total cholesterol,triglycerides by ELISA.

As depicted in FIGS. 13A and 13B, oligonucleotides targeting miR-122decreased target miRNA in vivo by 75-90% compared to PBS treatedcontrols. Monomers exhibited 75% knockdown of miR-122; whereas dimerscaused 90% knockdown of miR-122.

As depicted in FIGS. 14A and 14B, 50 mg/kg dose of oligonucleotidestargeting miR-122 decreased target miRNA in vivo by 90-95% compared toPBS treated controls. Monomers exhibited 90% knockdown of miR-122;whereas dimers caused 95% knockdown of miR-122. It was noted thatmonomer at 50 mg/kg is equivalent to dimer at 10 mg/kg for % miR-122knockdown.

As illustrated in FIG. 15, in vivo results show that dimers at 10 mg/kgexhibits similar knockdown as monomer at 50 mg/kg. Thus, dimeroligonucleotides are ˜5× more active than monomer (in vivo 7 d study).

MALAT-1 Study Results

Female Balb/c mice which were 7 weeks at shipment were evaluated (N=5).The mice were dosed at 50 mg/kg on Thursday, and takedown was at 5 dayspost-dose. Sample obtained from the mice included tserum, kidney, brain,and liver. Organs with high levels of MALAT-1 are heart, kidney, brainand minimally found in spleen and skeletal muscle. qRT-PCR was performedto evaluate Malat-1 knockdown. As depicted in FIGS. 16A-16C, dimeroligonucleotides robustly decreased Malat-1 lncRNA expression; where thecontrol GusB gene was unaffected.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1. A single-stranded nucleic acid compound comprising the generalformula: 5′X3′-L-5′X3′, wherein each X is independently asingle-stranded targeting oligonucleotide of 8 to 16 nucleotides inlength having a region of complementarity comprising at least 7contiguous nucleotides complementary to a target region of a genomictarget sequence, wherein adjacent nucleotides of the region ofcomplementarity of each X comprise phosphorothioate linkages, andwherein L is a linker consisting of 1 to 10 pyrimidine nucleotideslinked through phosphodiester linkages that links at least two Xs andthat is i) more susceptible to cleavage in a liver mammalian extractthan each X and ii) more susceptible to cleavage in liver mammalianextract than mammalian serum or plasma, and wherein when the targetregions complementary to the first X and second X do not overlap in thegenomic target sequence, the 5′-end of the target region complementaryto the first X and the 3′-end of the target region complementary to thesecond X are not within a distance of 0 to 4 nucleotides in the genomictarget sequence. 2-3. (canceled)
 4. The single-stranded nucleic acidcompound of claim 1, wherein the pyrimidine nucleotides are thymidinesor uridines. 5-64. (canceled)
 65. A composition comprising asingle-stranded nucleic acid compound of claim 1 and a carrier.
 66. Acomposition comprising a single-stranded nucleic acid compound of claim1 in a buffered solution.
 67. (canceled)
 68. A pharmaceuticalcomposition comprising a single-stranded nucleic acid compound of claim1 and a pharmaceutically acceptable carrier.
 69. A kit comprising acontainer housing the composition of claim
 65. 70-99. (canceled) 100.The single-stranded nucleic acid compound of claim 4, wherein thepyrimidine nucleotides are thymidines.
 101. The single-stranded nucleicacid compound of claim 4, wherein the pyrimidine nucleotides areuridines.
 102. The single-stranded nucleic acid compound of claim 1,wherein the target region of a genomic target sequence is a targetregion in an mRNA.
 103. The single-stranded nucleic acid compound ofclaim 1, wherein the target region of a genomic target sequence is atarget region in a long non-coding RNA (lncRNA).
 104. Thesingle-stranded nucleic acid compound of claim 1, wherein each targetingoligonucleotide comprises phosphorothioate internucleotide linkagesbetween all nucleotides.
 105. The single-stranded nucleic acid compoundof claim 1, wherein each targeting oligonucleotide comprises a lockednucleic acid (LNA) nucleotide, ethylene bridged nucleic acid (ENA)nucleotide, 2′-O-methyl nucleotide, or 2′-fluoro-deoxyribonucleotide.106. A method of delivering multiple targeting oligonucleotides to acell, the method comprising: contacting a cell with a single-strandednucleic acid compound of claim 1 under conditions in which the compoundenters into the cell.
 107. The method of claim 106, wherein the cell isin vitro.
 108. The method of claim 106, wherein the cell is in vivo.109. The method of claim 106, wherein the single-stranded nucleic acidcompound is delivered systemically to a subject comprising the cell.110. The single-stranded nucleic acid compound of claim 1, wherein thelinker consists of 2 to 4 pyrimidine nucleotides linked throughphosphodiester linkages.
 111. A compound comprising the general formula:5′X3′-L-5′X3′, wherein each X is independently a single-strandedtargeting oligonucleotide of 8 to 16 nucleotides in length having aregion of complementarity comprising at least 7 contiguous nucleotidescomplementary to a target region of a genomic target sequence, whereinadjacent nucleotides of the region of complementarity of each X comprisephosphorothioate linkages, wherein L is a linker consisting of 1 to 10pyrimidine nucleotides linked through phosphodiester linkages that linksthe Xs and that is i) more susceptible to cleavage in a liver mammalianextract than each X and ii) more susceptible to cleavage in livermammalian extract than in mammalian serum or plasma, wherein when thetarget regions complementary to the first X and second X do not overlapin the genomic target sequence, the 5′-end of the target regioncomplementary to the first X and the 3′-end of the target regioncomplementary to the second X are not within a distance of 0 to 4nucleotides in the genomic target sequence. and wherein the compounddoes not mediate degradation of the target nucleic acids by an RNAipathway.
 112. The compound of claim 111, wherein the pyrimidinenucleotides are thymidines or uridines.
 113. The compound of claim 112,wherein the pyrimidine nucleotides are thymidines.
 114. The compound ofclaim 112, wherein the pyrimidine nucleotides are uridines.
 115. Thecompound of claim 111, wherein the target region of a genomic targetsequence is a target region in an mRNA.
 116. The compound of claim 111,wherein the target region of a genomic target sequence is a targetregion in a long non-coding RNA (lncRNA).
 117. The compound of claim111, wherein each targeting oligonucleotide comprises phosphorothioateinternucleotide linkages between all nucleotides.
 118. The compound ofclaim 111, wherein each targeting oligonucleotide comprises a lockednucleic acid (LNA) nucleotide, ethylene bridged nucleic acid (ENA)nucleotide, 2′-O-methyl nucleotide, or 2′-fluoro-deoxyribonucleotide.119. The compound of claim 111, wherein the linker consists of 2 to 4pyrimidine nucleotides linked through phosphodiester linkages.